In a logistic regression analysis, Attention Deficit/Hyperactivity Disorder showed a significant association with maternal age  but not with vitamin D levels .We found no difference in intrauterine vitamin D levels between children later developing Attention Deficit/Hyperactivity Disorder and matched control children. However, the statistical power of the study was too weak to detect an eventual small to medium size association between vitamin D levels and Attention Deficit/Hyperactivity Disorder. Vitamin D deficiency is a common condition worldwide ,2. AlthoThe aim of the study was to test the hypothesis that low levels of vitamin D in the child during pregnancy are a risk factor for ADHD in the offspring.The selection procedure of children diagnosed with ADHD has previously been described . BrieflyUsing the personal identification numbers, umbilical cord serum samples for children with ADHD were retrieved from the Malmö Maternity Unit Biobank (MMUB). Almost all child deliveries in Malmö take place at the Skåne University Hospital Maternity Unit, where maternal and umbilical cord blood samples have been collected and stored in the MMUB. Maternal blood was collected by venepuncture, a few hours prior to delivery, and cord blood was collected by drainage into sterile sample tubes immediately after birth. The sample tubes were stored overnight in a refrigerator at +8°C for sedimentation. The following morning, the serum was collected and frozen in polypropylene plastic test tubes at -20°C until analysis.A pool of 10 eligible controls per child with ADHD were retrieved from the Swedish Medical Birth Register (SMBR)  and matc3 and the internal standard D6-25-hydroxyvitamin D3 were obtained from Toronto Research Chemicals, Inc. . In all analytical batches there were two different quality control (QC) samples prepared in- house and chemical blanks analysed. The control samples were checked against controls from Chromsystems Instruments & Chemicals GmbH  and the DEQAS Vitamin D External Quality Assessment Scheme . The samples were prepared and analysed in random order in triplicates and the mean value of the two closest concentrations was used in the statistical analyses. The variation coefficient for these analyses was 13%.Quantitative analyses of cord sera were conducted using a triple quadrupole linear ion trap mass spectrometer  coupled to a liquid chromatography system . Vitamin D concentrations were determined by a modified method for analysis of perfluorinated compounds . AliquotThe study protocol followed the requirements of the Declaration of Helsinki and was approved by the Research Ethics Committee at Lund University, Sweden (Reg.no. 2011/724)th and 25th percentiles of vitamin D levels among controls were arbitrarily chosen to define possible thresholds. Quartiles of vitamin D levels were also analysed. The possible associations between vitamin D levels below the thresholds and ADHD were tested using conditional logistic regression. Maternal data were analysed as risk factors for ADHD with conditional univariable logistic regression and variables with p values less than 0.20 were considered as potential confounders in a final multivariable conditional logistic regression analysis. The covariates analysed were maternal age, maternal smoking, season of birth, maternal BMI, parity and maternal height, see The Wilcoxon matched-pairs signed-rank test was used to compare the vitamin D concentrations between children with ADHD and matched control children. The Kruskal-Wallis/Wilcoxon test was used to compare vitamin D levels stratified for possible confounders. ANOVA analyses were performed concerning differences in the means for vitamin D in children with ADHD compared to control children, stratified for all cases and for children with a mother born in Sweden. A possible linear relationship between vitamin D levels and the risk of ADHD was investigated using logistic regression analysis. The odds ratio (OR) with 95% confidence interval (CI) was calculated. A spline analysis was performed which did not detect any possible threshold for the vitamin D–ADHD analyses. The 10th percentile was 1.8.With 202 children with ADHD and controls matched 1:1, and α = 0.05, the study had an 80% chance of detecting a difference of 0.3 SD. The lowest detectable OR for a value below the 25th vitamin D concentration percentile among controls was 9.17 ng/ml. No association between a value below this threshold and ADHD could be detected ; nor could an association between a vitamin D value below the 10th percentile (5.84 ng/ml) and ADHD be detected . The association between vitamin D values in the lower quartile among the Swedish controls (<10.4 ng/ml) and ADHD was also analysed, but was not statistically significant . Two seasonal periods had p-values lower than 0.20. When different maternal data  were analysed as risk factors for ADHD, only smoking and maternal age had p- values less than 0.20. In the conditional multivariable regression analysis, only maternal age and smoking had a p- value less than 0.20, so the final conditional logistic regression model was performed with maternal age, maternal smoking, two seasonal periods of birth and vitamin D levels as independent variables. Maternal age  and season of birth July to September  showed a significant association with ADHD , but not smoking , season of birth January to March  or vitamin D levels .The matching of the time of birth showed no heterogeneity between birth periods between cases and controls ), indicating only small differences in the storage time of the samples from cases and controls. Analyses stratified for sex, season of birth, maternal smoking and maternal BMI were performed, see Our study fails to support the hypothesis that low levels of vitamin D during pregnancy are a risk factor for ADHD in the offspring. No other study of vitamin D levels in children, with a prospective or pseudo-prospective design, has previously been performed. In a study from Qatar, vitamin D in serum was determined in 1331 ADHD children age 5–18 years and in the same number of controls. The ADHD children had lower levels than the controls . These rThis is a medium- sized study and a weak association with the risk of developing ADHD with vitamin D deficiency could not be excluded. The children with ADHD were diagnosed at the clinic for child and adolescent psychiatry, which means that the diagnoses are clinical and do not follow a research study protocol. The diagnoses were made more than ten years ago, and it is probable that the children diagnosed at that timed had more marked symptoms of ADHD than many have today, as the diagnostic criteria have changed towards a more liberal attitude. We have only analysed one single blood sample taken from the umbilical cord at birth from each child. This may not reflect vitamin D levels over time. In a study from 2013 by Major et al. of 583 adult individuals , single We found no support for the hypothesis that low levels of vitamin D during pregnancy are a risk factor for ADHD in the offspring. As a consequence of the limited statistical power, we cannot exclude that the may be a small to medium sized association between vitamin D levels at birth and a later diagnosis of ADHD.


ND) in patients with ADHD and healthy controls (HCs). Twenty adult patients with ADHD and 20 HCs underwent ‐[18F]FMeNER‐D2 positron emission tomography (PET) and were genotyped on a MassARRAY MALDI‐TOF platform using the Sequenom iPLEX assay. Linear mixed models analyses revealed a genotype‐dependent difference in NET BPND between groups in the thalamus and cerebellum. In the thalamus, a functional promoter SNP (−3081 A/T) and a 5′‐untranslated region (5′UTR) SNP (−182 T/C), showed higher binding in ADHD patients compared to HCs depending on the major allele. Furthermore, we detected an effect of genotype in HCs, with major allele carriers having lower binding. In contrast, for two 3′UTR SNPs , ADHD subjects had lower binding in the cerebellum compared to HCs depending on the major allele. Additionally, symptoms of hyperactivity and impulsivity correlated with NET BPND in the cerebellum depending on genotype. Symptoms correlated positively with cerebellar NET BPND for the major allele, while symptoms correlated negatively to NET BPND in minor allele carriers. Our findings support the role of genetic influence of the NE system on NET binding to be pertubated in ADHD. Hum Brain Mapp 37:884–895, 2016. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.Attention deficit hyperactivity disorder (ADHD) is a heterogeneous disorder with a strong genetic component. The norepinephrine transporter (NET) is a key target for ADHD treatment and the NET gene has been of high interest as a possible modulator of ADHD pathophysiology. Therefore, we conducted an imaging genetics study to examine possible effects of single nucleotide polymorphisms (SNPs) within the NET gene on NET nondisplaceable binding potential (BP Attention deficit hyperactivity disorder (ADHD) is the most frequent neurodevelopmental disorder diagnosed in children. It is characterized by inattention, hyperactivity, and impulsiveness which frequently leads to severe social, academic, and vocational dysfunction FMeNER‐D2. Moreover, we hypothesized that the symptoms of hyperactivity and impulsivity would correlate to with NET BPND in areas related to motoric activity  whilst symptoms of inattention would correlate with NET BPND in the thalamus.Thus, the aim was to examine the relationship between the effects of SNPs in the NE system and the NET BPTwenty adult ADHD patients  and 20 HCs  were recruited through ADHD outpatient clinic at the Department of Psychiatry and Psychotherapy, Medical University of Vienna, and from the local community via advertisement as previously published elsewhere FMeNER‐D2, synthesized as previously described FMeNER‐D2 is currently the most suitable radioligand for in vivo NET quantification previously described FMeNER‐D2. Mean specific radioactivity of ‐[18F]FMeNER‐D2 was 537 ± 383 GBq/μmol (ADHD patients) and 473 ± 218 GBq/μmol (HC) . Brain radioactivity was measured in a series of six consecutive time frames lasting 10 min each in the interval of 120–180 min after tracer bolus application. Acquired data were reconstructed in volumes consisting of 35 transaxial sections (128 × 128 matrix) using an iterative filtered back projection algorithm (FORE‐ITER) with a spatial resolution of 4.36‐mm full‐width at half maximum 1 cm next to the center of the field of view. For coregistration, magnetic resonance (MR) images were acquired from all participants on a 3‐Tesla Philips scanner (Achieva) using a 3‐D T1 FFE‐weighted sequence, yielding 0.88‐mm slice thickness and in plane resolution of 0.8 × 0.8 mm FMeNER‐D2 inherent to the radioligand. NET BPND for each region was extracted from parametric maps from the Hammers Maximum Probability Atlas FMeNER‐D2. Our results showed significant differences in cerebellar and thalamic NET binding dependent on genotypes between patients with ADHD and HCs. These were largely due to the impact of NET gene polymorphisms on NET BPND in HCs which is not as pronounced in patients with ADHD. Strikingly, in patients with ADHD, a high correlation between specific behavioural symptoms, that is, hyperactivity/impulsivity, and NET BPND in the cerebellum was detected, an effect which was strongly moderated by genotype.Here, we report the influence of genetic variants within the NE system and its effect on in vivo NET binding using PET and the radioligand  deviate from in vitro experiments which found that the minor (T) allele resulted in decreased promoter activity and the major allele (A) in higher expression FMeNER‐D2 cortical areas cannot be properly assessed due to spill over from suspected bone uptake. Therefore, genetic influence in the neocortex could not be examined. Studying the cortex, specifically the frontal cortex due to its vast role in cognitive and behavioral control would be very intriguing to test whether and what effects polymorphisms in the NET gene would have on the binding. Another limitation is that we did not include the CAARS inconsistency index and, therefore, we could not assess whether there was any inconsistencies or irregularities in responses within subjects, nor could we analyze that in respect to our data.A limitation of this study is that with (The evidence presented in this article gives rise to a role for genetic influence of the NE system and alterations in NE signalling as a part of the pathophysiology contributing to ADHD and thus strengthening the hypothesis of imbalances in NE system in the neurobiological mechanism of ADHD. However, we did not detect any differences in the midbrain and the putamen. We can only hypothesize about the possible reasons for these distinct effects. Our results may suggest that the NET genotype effects modulate the NET availability in a region specific manner. Conversely, we cannot exclude other possibilities, such as other influential genetic or nongenetic factors that interact with these regions. In addition, we did not detect any association of any SNP to ADHD, which is probably due to insufficient power of this sample to assess these subtle effects. Due to the heterogeneous nature of ADHD further research requires larger samples for the validation and for the establishment of potential endophenotypes in ADHD. Replication in vivo and in vitro studies is needed to establish if NET genotypic influence could serve as an endophenotype for NE neurotransmission. Moreover, SNPs within the NET may also be very important in relation to the dopaminergic system. The NET is also responsible for reuptake of dopamine in cortical regions [Morón et al., ND in patients with ADHD compared to HCs, depending on their genotype. We find genotypic difference in the thalamus between major and minor alleles for a functional promoter SNP in HCs only. The inverse effect of genotype which was detected in the cerebellum indicates genetic influence of NET on the binding in the cerebellum to differ between groups of ADHD subjects and HCs. The results are compatible with the theory that NE follows an inverted‐U‐shaped curve. Its effect on differential association of behavioral scales with binding further demonstrates a functional and neuropsychological activity to be imbalanced in ADHD.To conclude, this is the first imaging genetic study showing significant differences in NET BP


Bartonella henselae, a flea-transmitted bacterium, causes chronic, zoonotic, blood stream infections in immunocompetent and immunocompromised patients throughout the world. As an intra-erythrocytic and endotheliotropic bacterium, B. henselae causes a spectrum of symptomatology ranging from asymptomatic bacteremia to fever, endocarditis and death. Veterinary workers are at occupational risk for acquiring bartonellosis. As an emerging, and incompletely understood, stealth bacterial pathogen, B. henselae may or may not have been responsible for the deaths of two veterinarians; however, recent evidence indicates that this genus is of much greater medical importance than is currently appreciated by the majority of the biomedical community. Bartonella henselae contribute to the deaths of two veterinarians?” will never be definitively answered; however, after months of asking myself this question, I decided to comment on the possibility that a recently discovered stealth bacteria [The question “Did bacteria  may haveBartonella testing.Perhaps questions for which there are not definitive answers should never be asked, but if true, there would be no need for biomedical research. Unfortunately, many questions remain unanswered, a fact that is very hard for patients, family members, doctors, and diagnosticians to accept. Medicine remains “a practice,” based upon the best available science, the art of patient care, and a clinician’s attempts to manage symptoms and disease processes for which causation is elusive. Because of circumstances beyond everyone’s control, involvement of my research laboratory team was less than optimal due to the timing of specimen collection and type of samples available for st century patient care have advanced tremendously since Voltaire’s time; however, there are still major gaps in scientific knowledge that negatively impact patient outcomes. Specifically, our collective lack of “knowledge” negatively impacts directed therapy and effective management of the patient’s illness and deficiencies in medical knowledge related to the genus Bartonella continue to compromise patient care throughout the world.Voltaire, (1694 to 1778), stated: “Doctors put drugs of which they know little, into our bodies of which they know less, to treat diseases of which they know nothing at all.” Clearly, medical science and 21Bartonella henselae. The term “emerging infectious disease” has been abused and to an extent overused in recent years, often in association with efforts to influence the appropriation of research funding. During the past two decades, bartonellosis has clearly earned the designation “an emerging infectious disease.” The genus Bartonella has expanded from two known species prior to 1992 to at least 34 Bartonella species in 2015. Medically, fifteen Bartonella species have been associated with a spectrum of human illnesses; therefore, incompletely understood is another way to accurately describe this genus. The fascinating history of Bartonella has been summarized in recent reviews [Bartonella quintana or B. henselae in AIDS patients [Bartonella sp. had never been diagnosed by a physician or veterinarian in North America. Not surprisingly, clinicians cannot diagnose an as yet “unknown or undiscovered” infectious agent. Importantly, the history of Bartonella spp. infections substantially pre-dates AIDS. Bartonella bacilliformis, transmitted by sandflies, caused Oroya Fever and verruga peruana in indigenous Peruvian Indians hundreds of years ago; infected Spanish conquistadors, and caused highly fatal illness in immigrant workers building the Trans Peruvian railroad [Bartonella species, B. quintana, transmitted by the human body louse, was a major cause of morbidity and mortality during the World Wars [Bartonella species infections in AIDS patients, numerous new species have been implicated as a cause of illnesses in immunocompromised and immunocompetent patients [Bartonella [It is my hope that this commentary will benefit future patients, particularly veterinary workers, who have been or will become infected with the emerging and incompletely understood, stealth bacterial pathogen; patients , bartonepatients , 8. Desprtonella , there aDue to their lengthy illnesses, frequent medical evaluations, and involvement of numerous medical specialists and medical centers throughout the United States, only a brief, superficial overview of each veterinarian’s illness is possible. As an example of the medical complexity, veterinarian #1 was transferred among six different hospitals during the last 5 months of his life (October 2012 through February 2012). Selected historical and demographic information is summarized . This man was in good health until June 2010, when he lost consciousness, fell and was diagnosed with pneumonia. In April 2011, he had mitral valve replacement surgery, closure of a patent foramen ovale, 2-vessel bypass, bilateral maze procedure, radio frequency ablation, ligation of the left atrial appendage and pacemaker placement. Based upon self-reporting in a standardized questionnaire completed by all participants in our IRB-approved study, progressive symptoms including fatigue, disorientation, blurred vision, balance problems, difficulty remembering, eye pain, insomnia, muscle weakness, loss of sensation or numbness in the legs, and shortness of breath developed postoperatively in 2011. These non-specific symptoms are often reported by veterinary workers infected with one or co-infected with more than one ella sp. –14. Vetefections , 10. Becrtonella , 16. FroBartonella spp. are a cause of FUO and culture-negative endocarditis [et al. [Bartonella endocarditis was published in 1993. Subsequently, Bartonella endocarditis was reported in cats, cows, dogs and sea otters [Relapsing fever of unknown origin (FUO) with temperatures as high as 104 °F, developed in October 2011 (6 months after surgery). Despite various antibiotic therapies, febrile episodes, accompanied by periodic dizziness and increasingly severe muscle weakness, continued until his death on February 24, 2013. carditis –20. As r [et al. , the fira otters .Bartonella sp. isolation methods and PCR amplification of organism-specific DNA sequences from surgically removed or autopsy obtained heart valves have resulted in the identification of Bartonella endocarditis cases throughout the world [rd and January 30th, 2012, multiple blood cultures were obtained after consultation with an infectious diseases physician, of which a subset was sent to our laboratory for Bartonella testing. FUO had persisted despite prior treatment with cefixime and concurrent administration of doxycycline and ciprofloxacin. When Mycobacterium interjectum grew in one blood culture processed by a commercial laboratory, the patient was treated with doxycycline and clarithromycin beginning in March 2012, followed by clofazimine and clarithromycin. Mycobacterium interjectum, a slow growing mycobacteria most often associated with immunosuppression, generally responds to antibiotic treatment. Bartonella henselae (SA2 genotype) was amplified and sequenced from one of three blood specimens submitted to our laboratory , which he had been taking for most of the previous year for potential sarcoidosis. In January 2013, the patient was admitted to National Jewish Health, for evaluation of severe muscular weakness, dyspnea on exertion, memory loss and fever of unknown origin. During the diagnostic evaluations the patient’s condition deteriorated resulting in transfer to the University of Colorado in Denver. After flying home to Florida, the patient was hospitalized at Bayfront Medical Center, after which he was transferred to Shands Hospital, University of Florida. On February 13th, 2013, after 13 hours of surgery for vegetative valvular endocarditis, valve replacement surgery, splenectomy, and pacemaker removal the patient never regained consciousness. The death certificate listed multi-organ failure, sepsis, and mycobacterial endocarditis as the cause of death. Nearly two years after his death, B. henselae with sequence identity of 527/527 bp with B. henselae Fizz, (Gen Bank accession number AF369526) was amplified and sequenced from a paraffin-embedded bone marrow specimen obtained in January 2012. The same B. henselae Fizz genotype (identity 527/527 bp) was independently amplified and sequenced from the mitral valve prosthesis removed February 13, 2013, just prior to his death. Based upon blood and tissue PCR amplification and DNA sequencing results, this veterinarian was infected with two B. henselae genotypes in January 2012 and the Fizz genotype persisted at the time of his death. When and how these infections were acquired, how long the infections persisted, and if B. henselae contributed to his illness, FUO and ultimately his death will never be known.Microbiological improvements in he world , 18. BetBartonella research. Thus, it was a mutual friend who suggested Bartonella testing. Veterinarian #2 developed lymphadenopathy involving the neck, axillary lymph node (2 cm), and root of the mesentery . On February 24th, 2012, the left supraclavicular lymph node was surgically excised at Carolina East Medical Center. Histologically, the lymph node contained a mixed population of small and large lymphocytes, plasma cells, and scattered neutrophils, without evidence of necrosis, granulomatous inflammation, or abscess formation. There were no Reed-Sternberg cells indicative of Hodgkin’s disease. Preliminary pathological diagnosis was lymphadenitis of uncertain etiology. Fungal, bacterial and mycobacterial cultures were negative. Immunophenotypic studies, performed at Johns Hopkins Hospital as part of the standard lymphoma protocol, supported a diagnosis of angioimmunoblastic T cell lymphoma (AILT), with no specific morphologic or immunophenotpic characteristics to allow subclassification. In situ hybridization for Epstein Barr Virus (EBER) identified scattered positive cells. An addendum report from The Johns Hopkins Hospital contained the following statement: “Careful correlation with history is recommended to rule out immunodeficiency. The morphologic features coupled with a high proliferation rate and numerous mitoses are not seen in typical AILT cases; however, additional studies and clinical correlation are needed to exclude this possibility.” March 15th, 2012, the patient was referred to Duke University Medical Center where the pathologist concurred with the diagnosis of AILT, however, the attending lymphoma oncology specialist suggested the possibility of self-limiting lymphadenitis. July 17th, 2012, a pathologist at MD Anderson Medical Center concurred with the AILT diagnosis. A cervical lymph node, biopsied July 25th, had effacement of normal nodal architecture by atypical lymphoid infiltrates, accompanied by mild vascular endothelial proliferation, and scattered eosinophils and plasma cells. Angioimmunoblastic features were less prominent and features of lymphoma were more prominent than the previous biopsy. EBER staining was negative. Immuno-histochemical staining again supported a diagnosis of AILT. The cancer was PCR positive for clonal T-cell receptor gamma gene rearrangement and negative for clonal B-cell immunoglobulin gene rearrangement. Bartonella henselae can cause FUO [I have been a veterinary internist at North Carolina State University, College of Veterinary Medicine since 1982. As such, I consult with regional veterinarians on a near daily basis. Despite the fact that Veterinarian #2 practiced medicine in North Carolina for 37 years, I did not know him personally; however, as fate would have it, he lived in a house along the North Carolina coast adjacent to a close friend who was familiar with our ause FUO , 19.Bartonella testing. Because of the historical administration of several antibiotics, BAPGM enrichment blood culture/PCR was likely to be diagnostically less sensitive; therefore, paraffin-embedded lymph node biopsies obtained in February and July 2012 were tested by conventional PCR. Bartonella henselae (SA2 genotype) was successfully amplified and sequenced from the July lymph node specimen. Shortly thereafter, treatment with doxycycline as the sole therapy was instituted for bartonellosis and the rigors and fevers temporarily resolved. In completing the study questionnaire, this veterinarian reported being healthy prior to the development of lymphadenopathy in February 2012, and with the exception of FUO accompanied by rigors and night sweats, CHOP  chemotherapy had been well tolerated. When the B. henselae lymph node PCR result became available, I related our experience with doxycycline therapy in experimentally and naturally infected cats [Bartonella sp. bacteremia, but rarely, if ever, cured the infection. In humans, doxycycline treatments as long as 3 months in duration have resulted in symptomatic and hematological improvement, but sequential blood culture/PCR indicated B. henselae infections persisted [By September 2012, Veterinarian #2 was referred to MD Anderson Center. He was anemic , and had a lymphocytosis . A core bone marrow biopsy contained multiple, atypical, lymphohistiocytic aggregates, with a prominent epithelioid component. Immunophenotyping supported AILT with marrow involvement. In October 2012, Veterinarian #2 requested ted cats , and natted cats  to the persisted , 23.B. henselae infection was not detected by BAPGM enrichment blood culture/PCR [st, 2013. The planned allogeneic bone marrow stem cell transplantation was never performed.Due to the progressive nature of the AILT, a decision was made to treat the patient with allogeneic bone marrow stem cell transplantation; however, this therapy was repeatedly delayed between October 2012 and April 2013 due to recurrent bouts of FUO, which were temporally followed administration of chemotherapy. ture/PCR  in AprilB. henselae contribute to the deaths of these two veterinarians?”; we would first have to ask, does B. henselae cause FUO and endocarditis? The answer is yes. A second important question is: “What microbiological evidence supports B. henselae infection?” For Veterinarian #1, B. henselae DNA was amplified and sequenced from three diagnostic specimens  collected at three different time points between January 2012 and February 2013. For Veterinarian #2, B. henselae DNA was amplified and sequenced from a paraffin-embedded lymph node obtained surgically, four months prior to PCR testing in our laboratory. Subsequently, despite ongoing febrile illness, serology and BAPGM enrichment blood culture testing failed to identify B. henselae DNA or antibodies. As summarized in a recent review [Bartonella spp. DNA contamination with every sample set processed within the laboratory. DNA carryover and amplicon contamination are considered an unlikely source for the microbiological findings in these two veterinarians. Although isolation is the microbiological “gold standard” for documenting a bacterial infection, PCR amplification of organism-specific DNA sequences is being used to document the presence of fastidious [B. henselae infection in both veterinarians.In asking the question: “Did t review , our rest review , 27. We t review . Our labstidious , stealthstidious . PCR ampB. henselae?” For reasons that remain unclear, a substantial (50-75 %) subset of Bartonella spp. bacteremic patients do not have detectable IFA antibodies [Bartonella bacteremia, a seronegative status may be the norm, rather than the exception; thus, serology is not a diagnostically or epidemiologically sensitive modality. Often after weeks or months of prior antibiotic therapy, many veterinary workers are tested for Bartonella infection as an afterthought or as a “test of last resort.” Using currently available diagnostic techniques, these bacteria are difficult to enrich, to isolate or to PCR amplify from tissues under optimal testing conditions. Long delays before obtaining or processing patient specimens, concurrent or recent antibiotic administration, and the number of blood or tissue samples tested influence the sensitivity of the BAPGM enrichment blood culture/PCR platform. [Bartonella microbiological findings, than we reported above.Another important microbiological question; “Was there serological evidence to support exposure to tibodies , 31. Witlatform. , 32 For latform. , 27 are Bartonella bacteremic veterinary workers [Bartonella sp. bacteremia in “non-immunocompromised” individuals is not impossible, as we have repeatedly demonstrated [B. henselae infection in immunocompetent people is synonymous with cat scratch disease (CSD), which is considered a self-limiting infection. Although CSD is most often self-limiting, this does not appear to be uniformly true [B. henselae bacteremic patients with associated symptomatology and allow for the documentation of progression of rheumatologic [Assuming the possibility that both veterinarians were infected with viable bacteria at the onset of illness and/or when tested, the next question becomes: “Did the bacterium play a role in the initiation or progression of each patient’s illness?” For Veterinarian #1, the approximate two-year history of non-specific, waxing and waning symptoms is consistent with the questionnaire responses reported by other  workers –14. Estanstrated –14, 23. mly true . Performmly true , might iatologic  and/or natologic .B. henselae infection predispose to the development of lymphoma?” Bartonella spp. can invade numerous host cells, within which the bacteria modify cellular functions by injecting peptides and potentially transporting bacterial DNA into the cell [B. henselae contains bacteriophages [Bartonella spp. appear to play causative or cofactor roles in the development of vasoproliferative tumors in animals [Bartonella vinsonii subsp. berkhoffii became immunosuppressed [B. bacilliformis [A biologically more complex question is: “Did the cell , 6, 34. iophages  that mig animals  and immu animals . Althougppressed . Infectiliformis , 4. PoteB. henselae play a role in the deaths of two veterinarians? Perhaps yes and perhaps no. For both veterinarians, it seems likely that B. henselae was responsible for FUO. During their illnesses, and after their respective deaths, I communicated with both wives. Based upon their comments during these challenging conversations, it is their hope that some good may come from their husband’s deaths. Both men were outstanding fathers, husbands, veterinarians and community servants, each participating in a large number of volunteer activities. From my perspective, it is time for a “Bartonella tipping point,” whereby this emerging pathogen is accorded a higher national (United States) and international research priorities.Did


Widespread disparities in white matter (WM) microstructure and organization have been found in adolescents with attention-deficit/hyperactivity disorder (ADHD); however, little is known about the role sex plays in these differences. The present diffusion tensor imaging (DTI) study performed whole-brain, tract-based, voxel-wise, and region of interest (ROI) analyses to investigate WM microstructure differences between ADHD and healthy control (HC) adolescents to examine the impact of sex on measures of fractional anisotropy (FA). Eighteen adolescents with ADHD and 24 HC were included in this study. All participants received a 64-direction DTI scan on a 3 Tesla Siemens scanner. FSL's TBSS was used to perform whole-brain, tract-based, voxel-wise analyses. Tracts demonstrating significant sex-by-diagnosis interactions were further evaluated using univariate analyses performed on mean FA data that were extracted from ROIs using the Johns Hopkins University WM tractography atlas. TBSS analyses between diagnostic groups revealed significantly increased FA in HC relative to ADHD in the bilateral superior longitudinal fasciculus (SLF), forceps major, left cingulum, and bilateral callosal regions. In addition, both TBSS and separate ROI analyses revealed significant sex-by-diagnosis interactions for the corticospinal tract (CST), inferior longitudinal fasciculus (ILF) and SLF. In the HC group, FA was increased in males relative to females for all analyses. In WM regions demonstrating a significant sex-by-diagnosis, FA was increased in females relative to males in the ADHD group. Our findings suggest that WM microstructure in several major WM tracts differs between males and females with ADHD. These differences in WM microstructure may account for some of the differences in ADHD subtypes and comorbidities seen between the sexes. Additional studies in ADHD, examining sex differences in phenotypic expression, treatment response and brain network trajectories are warranted. Attention deficit/hyperactivity disorder (ADHD) is one of the most frequently diagnosed chronic neurodevelopmental disorders in children and adolescents with recent reports citing prevalence rates of up to 10% for United States (US)  is a magnetic resonance technique used to investigate WM microstructure. The microstructure and organization of WM tracts is often reported using fractional anisotropy (FA), which is a measure of the degree of anisotropic diffusion of water molecules in brain tissue, which can be affected by cellular structure. Areas of high fiber organization where axons are more heavily myelinated, such as the corpus callosum, tend to have higher FA values. This imaging technique has been applied to the study of ADHD where differences in FA values have been found in ADHD relative to HC, though the directionality of these variances have been inconsistent  measurements of DTI FA data to determine if differences existed between diagnostic groups as well as if sex indeed impacts WM microstructure differently between male and female youths with ADHD as compared to HC. We hypothesized that sex differences in WM microstructure in youths with ADHD would differ from that of HC youths. More specifically, we predicted that these differences would manifest as increased FA in females with ADHD as compared to males due to evidence suggesting earlier WM tract maturation in females with ADHD. Furthermore, we examined associations between WM integrity and measures of clinical symptoms often associated with ADHD including inattention and impulsivity/hyperactivity.The Institutional Review Board at the University of Utah approved this study. All subjects provided written informed assent prior to study participation. Parental consent was also acquired for all study participants. Recruitment efforts included the use of local advertisements and word of mouth. A total of 43 subjects, including 19 youths with ADHD  and 24 HC youths , were included in this analysis. Inclusion criteria for study participants were: male or female, 10–18 years of age, and of any race or ethnicity. A clinical and diagnostic semi-structured interview (Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present an Lifetime Episode (K-SADS-PL)  . Original images were transferred from the scanner in the DICOM format and coded. Participants' MRI scans were read by a neuroradiologist to rule out gross pathology.Acquisition of imaging data was performed at the Utah Center for Advanced Imaging Research (UCAIR) using a 3T Siemens Trio scanner. A DT-MRI GRAPPA sequence was obtained utilizing 64 directions, 2 diffusion weightings: www.fmrib.ox.ac.uk/fsl). In depth details and description of the DTI processing stream can be located on the FSL website. Briefly, images were first corrected for motion and eddy distortion; next, non-brain tissue was removed using BET  for Mac OS X. Independent student's n = 1), current oppositional defiant disorder (n = 2), and current depression (n = 1). Of the 19 ADHD study participants, 68.4% were currently taking stimulant medications at the time of the study; and of the 31.6% who were not, 50.0% of those individuals had a history of stimulant pharmacotherapy. Four ADHD participants (21.1%) were currently taking psychotropic medications other than stimulants .Participants in the ADHD group were diagnosed with either ADHD combined subtype (52.6%) or inattentive subtype (47.4%). Of the 19 participants with ADHD, 16 had a familial history of ADHD. Familial history data was not available for one study participant due to adoption. Four of the ADHD participants (21.1%) had been diagnosed with one past or current comorbid diagnosis including a history of depression (p < 0.001). Symptoms of impulsivity/hyperactivity and inattention as well as internalizing and externalizing symptoms, as assessed by the ARS and CBCL, respectively, were found to be significantly increased in ADHD when compared to sex matched HC . Male and female participants in the ADHD groups were similar in age, Tanner stage, FSIQ, GAF, ARS impulsivity/hyperactivity, ARS inattention, and CBCL internalizing and externalizing t-scores. In addition, male and female participants in the ADHD group were similar on current and past exposure to stimulant medications.Diagnostic groups were matched for sex; however, age was found to be higher in HC compared to ADHD     with increased FA in males relative to females in the HC group, while FA was increased in females relative to males in the ADHD group.Whole-brain, tract-based, voxel-wise comparisons revealed significant main effects of diagnosis by way of increased FA in HC relative to ADHD in the bilateral SLF, forceps major (FMA), left cingulum (cingulate gyrus) (CGC), and bilateral callosal regions p ≤ 0.0  = 4.11, p = 0.05] SLF-T. No regions indicated significantly greater FA in ADHD when compared to HC.For each study participant, mean FA values were extracted from masks within WM tracts identified in the whole-brain, tract-based, voxel-wise comparison. Univariate analyses, using diagnosis, sex, and sex-by-diagnosis as fixed factors and age as a covariate, were conducted on extracted FA values. Between diagnostic group analyses revealed significantly increased FA in HC relative to ADHD in the right [Significant sex-by-diagnosis interaction effects were found for the right CST, ILF, SLF, and SLF-T .Correlations with hyperactivity/impulsivity and inattention scores as well as externalizing and internalizing symptom scores were performed on extracted FA values in tracts that had demonstrated significant sex-by-diagnosis interaction effects for the ADHD group, i.e., CST, ILF, and SLF/SLF-T. Correlations were also evaluated for each sex within the ADHD group. For the total ADHD group and in the male subgroup, no significant correlations were found with any clinical measure. However, females with ADHD demonstrated a significant positive correlation between mean FA in the right ILF and ARS impulsivity score  when compared to age- and gender-matched HC (n = 15) in the left middle cerebellar peduncle, cerebellum, and parieto-occipital, and right cerebral peduncle, premotor, and striatal areas. Similarly, Nagel et al. (n = 16) in frontoparietal, frontolimbic, cerebellar, corona radiata, and temporo-occipital regions when compared to HC (n = 20). Hamilton et al. (n = 17), but in an all male sample, in the CST and SLF when compared to male HC (n = 16). Moreover, in a study comparing WM fiber tracts in youths with ADHD (n = 13), bipolar disorder (BPD) (n = 13), and HC (n = 15), Pavuluri et al.  and identified comorbidities (higher rates of anxiety) often seen in females with ADHD  and NIH: R01 DA020269 (DYT).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Symptoms of attention deficit hyperactivity disorder (ADHD) and autistic spectrum disorder (ASD) often co-occur. The DSM-IV had specified that an ASD diagnosis is an exclusion criterion for ADHD, thereby limiting research of this common clinical co-occurrence. As neurodevelopmental disorders, both ASD and ADHD share some phenotypic similarities, but are characterized by distinct diagnostic criteria. The present review will examine the frequency and implications of this clinical co-occurrence in children, with an emphasis on the available data regarding pre-school age. The review will highlight possible etiologies explaining it, and suggest future research directions necessary to enhance our understanding of both etiology and therapeutic interventions, in light of the new DSM-V criteria, allowing for a dual diagnosis. In the last decade, studies have reported increased prevalence of both attention deficit hyperactivity disorder (ADHD) and autistic spectrum disorders (ASD). While ADHD is defined by impaired functioning in the areas of attention, hyperactivity, and impulsivity, whereas ASD is characterized by core social dysfunction and restrictive-repetitive behaviors, studies show that between 30 and 50% of individuals with ASD manifest ADHD symptoms (particularly at pre-school age), and similarly, estimates suggest two-thirds of individuals with ADHD show features of ASD  and ADHD in children.” More than 150 articles were eventually reviewed.n = 312) from the Boston University Twin project were studied by Ronald et al. (r = 0.23–0.26), a lower correlation than described for older children. In a recent survey by Carlsson et al. .This profile of social difficulties differs, however, from that observed in ASD, in which impairment in the basic understanding of social realm is central, such as a lack of emotional reciprocity and engagement with others, and the low interest or enjoyment in social interaction. Cantwell  describeRecent research suggests many individuals with ADHD may experience social impairments consistent with those observed in ASD. Santosh and Mijovic  characteDiagnostic constraints have limited research on co-occurring ADHD and ASD, because many studies, in accordance with the DSM-IV, have excluded individuals with other psychiatric or developmental difficulties . In another study by Gadow et al.  deficits are common in both disorders, together with response inhibition deficit. EF measures hardly discriminated between ADHD and HFA, but compared to children with ADHD, the HFA group showed more difficulty with cognitive flexibility and planning , using parent and teacher questionnaires. Phenotypic correlations were 0.54 for parent and 0.51 for teacher data. The authors concluded that genetic correlations between ASD and ADHD symptoms were all >0.50. Similar results were reported in a study of young adult twins (N = 674), using a self reported measures of ADHD and ASD. A bivariate model indicated that the genetic correlation between ADHD and ASD was 0.72. Models with additive genetic effects and unique environmental effects fitted the data best, with no evidence for sex differences. While the limitation of most of these studies is the use of rating scales rather than direct clinical measures to evaluate either ADHD or ASD, together they do point at a genetic correlation between the two diagnoses.In a twin study, mentioned earlier  have specifically addressed this co-occurrence, pinpointing to some promising pleiotropic genes, loci, and single nucleotide polymorphisms (SNPs). Their authors comment that there is an urgent need for better designed and powered studies to tackle this complex issue. A recent study  for children with ADHD, there are only a few studies on the combined medication and behavioral approach in ASD children. Aman et al.  primarilStudies on psychosocial interventions in pre-school children with co-occurring ASD and ADHD are lacking.Research specifically focusing on co-occurring ADHD and ASD has only recently evolved, being previously limited by the DSM-IV exclusion criteria. What we have so far learned is that both disorders frequently co-occur, and when they do, they cause greater morbidity, and create a more complicated clinical challenge. The new DSM-V, allowing for a dual diagnosis, will hopefully facilitate research, by eliminating the exclusion of many patients and allowing the study of broader phenotypes. Most recent research focuses on etiology and clinical presentations, with less direct work on treatment and early intervention protocols. Very few studies have looked at pre-school children presenting with both conditions, on the impact of early intervention in this age group and its effect on developmental trajectory. Furthermore, many of the above quoted studies have used parent and/or teacher rating scales to assess clinical profiles, rather than direct clinical diagnoses of the two co-existing disorders. In the future, as a dual diagnosis is “officially” possible, studies using direct clinical assessment of both comorbid diagnoses are essential.Future research should tackle two major hypotheses regarding the frequent co-occurrence of ASD and ADHD. The first is that ADHD and Autism are distinct, yet overlapping disorders which may share some common etiology, probably genetic. The second hypothesis is that the co-occurrence of autistic symptoms and ADHD “stands alone” as a distinct clinical disorder, with a distinct etiology, and a different developmental trajectory. These two hypotheses should be examined by studying the developmental trajectories in co-occurring ASD and ADHD, by defining common co-morbidity profiles in both, and by understanding differences and similarities in social perception, motor functions and language, cognition, and EF in each disorder and in the co-occurring “phenotype.” Defining early “endophenotypes” as suggested by Rommelse et al.  of both disorders, should serve to increase the chance of identifying genetic markers for each one and for both together (Cross-Disorder Group of the Psychiatric Genomics Consortium, In 2010, Gillberg et al. have coined the ESSENCE concept (Gillberg, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Attention deficit hyperactivity disorder (ADHD) is a neglected illness in a developing country. Objectives. The objectives of this study are to investigate the prevalence and pattern of ADHD among children in a Nigeria. Methods. A structured self-administered questionnaire was used to collect information from the parents of children (and older children) who attended children outpatients' clinic during the study period. The DSM-IV-TR diagnostic criteria for attention deficit hyperactivity disorder were used. Results. Two hundred and seventy-three (273) out of 282 questionnaires were filled completely, giving a response rate of 96.8%. Nine (9) children fulfilled the stated criteria for ADHD giving a prevalence rate of 3.2%. There is no association between gender and ADHD (P = 0.784). Conclusions. The prevalence of ADHD in our setting is 3.2%, which is similar to that obtained elsewhere in the world. ADHD-PI or ADHD-I), predominantly hyperactive-impulsive (ADHD-HI or ADHD-H), or a combination of these two subtypes (ADHD-C) [Attention deficit hyperactivity disorder (ADHD) is defined by features of inattention, overactivity, and impulsivity. It is the most frequently encountered childhood-onset neurodevelopmental disorder in the primary care settings . Symptom(ADHD-C) . ADHD ha(ADHD-C) , 4.The prevalence of ADHD is rare in the published literature in Nigeria. And ADHD prevalence seems to vary in different settings, such as in the general population versus in hospitals or in schools. Not much is known about ADHD prevalence in hospitalized Nigerian children. And there are few documented findings about the prevalence of ADHD in the outpatient setting. In other African countries, such as South Africa, Democratic Republic of Congo, or Ethiopia, the prevalence of ADHD has been reported to vary from 5.4% to 8.7% among school children. However, in the general population, ADHD has been reported in 1.5% of children. And children with possible organic brain pathology have been reported to have a prevalence of ADHD of 45.5–100% .The prevalence of ADHD reported on other continents is variable. For instance, prevalence of ADHD in Saudi Arabian primary schools is reported to be as low as 2.7%, while that in Iran is reported to be as high as 13% , with a ADHD normally affects preschool age children, although it can extend beyond childhood and adolescence into adulthood . A higheLinnet et al. noted an unconfirmed link between socioeconomic factors and ADHD in children . This waThis study investigated the prevalence and pattern of ADHD in a developing country. Our hope is that this study will shed light on ADHD in a hospital setting, to help parents of affected children cope with a diagnosis of ADHD. Studies on the topic of ADHD in a developing country are available from the continent. Such studies are essential for the formulation of policies on intervention and delivery of healthcare services. The few studies that have already been conducted in Africa have mainly assessed children in a school environment. Unfortunately, many clinicians do not consider the fact that ADHD can also be diagnosed in children visiting the hospital. Many children come to the hospital, where they are diagnosed with malaria, other infections, and other common childhood illnesses. But, parents, caregivers, and even physicians sometimes miss a diagnosis of ADHD in this patient population. Thus, it may be useful to establish an area of paediatric outpatient clinics that is staffed by a child psychiatrist who can diagnose ADHD. This study will help to determine if there is a difference in the prevalence of ADHD diagnosed in a hospital versus a school setting. In addition, this study will establish baseline patterns of distribution of ADHD and will assist physicians who have a high index of suspicion to diagnose ADHD.This study was carried out in the paediatric outpatient clinic of two teaching hospitals in the Southeastern state of Nigeria: University of Nigeria Teaching Hospital (UNTH) Ituku-Ozalla, Enugu and Enugu State University Teaching Hospital (ESUTH) (both in Enugu State).Two hundred and seventy-three children and their parents who were seen on the paediatric wards of two teaching hospitals and who met inclusion criteria were consecutively recruited into our prospective study between the 1st of March and the 30th of June, 2013. The average number of patients seen was 50 per day at ESUTH and 40 per day at UNTH. However, only subjects who signed consent were included in this study.Z  = 1.96; P = Prevalence of children with ADHD 16% ; D = Tolerable error (0.05).The minimum sample size required for this study was calculated using the following formula :(1)N=Z2Using the formula above, we determined a minimum sample size of 206 patients. In anticipation of a 10% rate of attrition, the minimum sample size for our study is 270.A structured self-administered questionnaire was used to collect information from the parents of children, as well as older children, who presented to paediatric outpatient clinics during the study period. In a few cases in which the parents were illiterate, the questionnaire was administered by study investigators. Parents were asked to recall symptoms, from a list of criteria for the diagnosis of ADHD, exhibited by their children either at home or at school. We used the DSM-IV-TR diagnostic criteria for ADHD. According to these criteria, six of the nine symptoms in each section of inattention and hyperactivity/impulsivity must be present for at least 6 months to make a diagnosis of “combined type” ADHD [The paediatrician also performed a thorough physical examination of each child in the study over the course of the study. Parents of children who signed consent and who understood the questionnaire thoroughly were included in this study, while individuals with other medical or psychiatric disorders and children whose parents did not sign consent were excluded.Each family was assigned a socioeconomic class using a recommended method, modified by Oyedeji . ParentsIt is interesting and important to investigate the prevalence of ADHD in a hospital setting because a child may actually be hyperactive with other illnesses like malaria. The physician may, thus, ignore the former disorder and focus on the later. Thus, many children come to the hospital with various degrees of hyperactivity and inattention but go unnoticed and undiagnosed.Ambuabunos et al. [The questionnaire used in this study is a guide for the diagnosis of ADHD, adopted from the Disruptive Behaviour Disorder Rating Scale contained in the DSM-IV . This qus et al.  in BeninDuring the process of informed consent, parents or caregivers and children were informed that study participation was voluntary, that responses to the questionnaire were anonymous, and that they could withdraw from the study at any point in time if they so desired without any consequences.This study was designed to determine the prevalence of ADHD among children presenting to Nigerian hospitals and to determine if this prevalence was different from the known school prevalence. We also aimed to determine if there was a correlation between ADHD and socioeconomic class.Ethical clearance for this study was sought from the Research and Ethical Committee of the University of Nigeria Teaching Hospital Ituku/Ozalla, Enugu. Informed consent was sought from parents/caregivers of potential subjects before enrolling them into the study.Subjects who fulfilled the inclusion criteria were consecutively enrolled into the study.The objectives of this study are to investigate the prevalence and pattern of ADHD in our environment.t-test were used for categorical and continuous variables, respectively. The confidence interval was set at 95%.Data was analysed using the SPSS statistical package, version 17. The chi-square statistical test and P = 0.72). The age of study participants ranged between two- and 13-year-old, with mean age of 7.06 years old (P = 0.21).Two hundred and seventy-three out of 282 questionnaires were filled-out completely, which equated to a response rate of 96.8%. There were a total of 138 males and 144 females. This equated to a male to female ratio of 1 : 1 . Among those with ADHD, eight were predominantly inattentive and one was predominantly hyperactive.P = 0.46).Although ADHD criteria were fulfilled more often among children >6 years old than among those <6 years old, the association between ADHD and age was not significant (P = 0.6).From this study, we determined that the prevalence of ADHD was 3.2%. Several reviews of the literature have reported highly variable prevalence rates for ADHD worldwide, ranging from a low of 1% to a high of almost 20% among school-age children , 20. ThiIt has also been argued by other experts that the variability in ADHD/HD prevalence estimates may be best explained by the use of different case definitions. They argue that the actual prevalence across geographic sites should not vary when case definitions are identical , 27. IntIn the present study, we also found that ADHD prevalence does not vary by gender. This is different from the results of most studies where a male preponderance has been noted . AlthougIt is noted from this study that almost half of the children with ADHD were diagnosed before the age of six years old. In other studies, symptoms of ADHD may have been diagnosed before the age of six years old. And there were cases noted of late-onset ADHD in some studies. ADHD may affect not only children less than 6 years old, but even those up to 18 years old .In our study, we had more children with ADHD-I than with ADHD-H or ADHD-C. This was confirmed by Erik in his study where he noted a high frequency of ADHD-I among children with ADHD . This waAlthough children diagnosed with ADHD were noted to come mainly from middle and low socioeconomic classes, when we correlated socioeconomic class and prevalence of ADHD, we did not find an association between socioeconomic class and ADHD. Studies in some countries have indicated that individuals from low socioeconomic class environments are 1.5–4 times more likely to meet criteria for ADHD than individuals from families from high socioeconomic classes . HoweverWe conclude that the prevalence of ADHD in our setting was similar to that in other parts of the world, and there is need for additional studies in this subregion. This is because ADHD is one of the emerging mental health problems and a neglected health issue in Africa.


Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by symptoms of inattention, hyperactivity, and impulsivity. Soft neurological signs (SNS) are minor neurological abnormalities in motor performance, and are used as one evaluation method for neurodevelopmental delays in children with ADHD. Our aim is to establish a quantitative evaluation system for children with ADHD. We focused on the arm movement called pronation and supination, which is one such soft neurological sign. Thirty three children with ADHD aged 7–11 years  and twenty five adults participants aged 21–29 years old  participated in our experiments. Our results suggested that the pronation and supination function in children with ADHD has a tendency to lag behind that of typically developing children by several years. From these results, our system has a possibility to objectively evaluate the neurodevelopmental delay of children with ADHD. The current diagnostic methods for neurodevelopmental disorders are based on pre/perinatal, developmental, family history, and physical/neurological examination, including visual inspection of so-called soft neurological signs (SNS). SNS are minor neurological motor performance abnormalities used as findings to test for developmental delays in neurological function . SNS havPrevious studies have reported the differences between typically developing (TD) children and several developmental disorders: attention deficit hyperactivity disorder (ADHD) ,6,7,8,9,We focused on pronation and supination which is one of the SNS test methods. Previous studies have quantitatively measured this test ,16,17, hIn our previous paper, we reported a quantitative evaluation system using acceleration and angular velocity sensors . We measIn this study, our aim was to establish a quantitative evaluation system of soft neurological signs for children with ADHD. For this purpose we focused on ADHD which is one such developmental disorder. We measured 33 children aged 7–11 years with ADHD  and 25 adults aged 21–29 years  and looked into the characteristics of ADHD. Our system is comprised of four acceleration and angular velocity sensors , a guide monitor  and a notebook PC . The subject were attached these sensors on both hands and elbows, as shown in In accordance with observation items for developmental disorders, in this research we focused on two motor tasks: an imitative motor task and a maximal-effort motor task performed with only one hand. Measurement time was 10 s for each task. When the participants performed the imitative motor task, they imitated the motion on a guide monitor. The hands on guide monitor test was performed at a rate of 80 times per minute (about 1.3 Hz). In the maximal-effort motor task, the participants moved their hand as fast as possible. The participants were instructed to maintain their non-rotating hand in a horizontal position, as shown in Thirty three children with ADHD aged 7–11 years  and twenty five adults participants aged 21–29 years old  participated in our experiment . ChildreAll children and their parents received an explanation of the aim, and the procedures and hazards of the experiment before our measurement. They all agreed to participate. The participants attended the first experimental session. The study was approved by the Kyushu University Ethics Committee.There are four observation indices to evaluate pronation and supination: (a) speed; (b) associated movement; (c) elbow excursion, and (d) pauses at extreme positions of the pronated and supinated hand ,2. AssocRotational speed used the peak frequency of acceleration on the Z axis in the continuous fast Fourier transform (FFT). Mirror movements used the absolute value total sum of acceleration and angular velocity on the Z axis and X axis. Postural stability of rotating elbows was calculated using the absolute value total sum of acceleration on the Z axis. Measurement time per a task is 10 s. In this study, we separated seven phases in the measurement waveform  to analyze the temporal changes of measurement time. Therefore the value of a parameter was mean value of seven phases. Temporal change of rotational size is an index to evaluate whether the child can accurately pronate and supinate at a fixed rotational angle. Temporal change of rotational size used the variance in peak frequency’s power of the continuous FFT among the seven phases (10 s).Moreover, to compare between our previous results of typically developing children and data of children with ADHD, we used other two indices: bimanual symmetry and compliance. Bimanual symmetry is an evaluation index of whether the participant’s left hand and right hand move in symmetry or not. This index used the correlation coefficient of acceleration on the Z axis, and phase difference of acceleration waveform on the Z axis between the both hands. Compliance is an evaluation index of whether the motion speed between the subject’s hands and the hands on the guide monitor is the same or not. This index used the difference between about 1.3 Hz  and the rotational speed of the subject’s hands. The sampling frequency was 100 Hz. A 6 Hz low-pass was applied to the signal.hx is the analyzed value in all participants. The aμ and aσ are the mean value and standard deviation of the each analyzed values in participants aged 21–29 years old. The hy indicate score of pronation and supination in each indices. To compare between TD children and children with ADHD in same age group, we used the Mann-Whitney U test for statistics. Analyzed parameters discussed in the above were normalized by the data of participants aged 21–29 years old using the following formula:In our previous study, we could obtain the developmental curves of pronation and supination in TD children using our evaluation system. Therefore we looked into the characteristics of children with ADHD and the difference between TD children and children with ADHD by age in this study.In the one hand maximal-effort motor task, we have three evaluation indices: rotational speed, mirror movement and postural stability of elbow. As shown In case of TD children, the score increased as they grew older. In rotational speed and postural stability of rotating elbow, the score of ADHD increased the same as TD children with age. On the other hand, there was no relationship between age and score of mirror movement in children with ADHD. In the imitative motor task, we have three indices: bimanual symmetry, compliance and temporal change. As shown In bimanual symmetry, the score of children with ADHD increased with age. On the other hand, the variability of ADHD scores was larger than the variability of TD children’s score in compliance and temporal change of rotational size. There was no relationship between age and the score of children with ADHD in these indices. Pronation and supination is SNS test for children of 4 years and older. The function of pronation and supination improves as children grow. Mirror movement during pronation and supination is movement which manifests itself in the contralateral hand by associated pronation and supination . This moOn the other hand, the function of pronation and supination was lower than the function of TD children. In rotational speed, postural stability of rotating elbow and bimanual symmetry, the pronation and supination function in children with ADHD has a tendency to develop several years late. Moreover, the function’s balance of children with ADHD is similar to the function’s balance of TD children who are younger than the age of the children with ADHD. Previous studies reported that cortical development of children with ADHD lagged behind that of TD children by several years . Our resHowever, there was no relationship between age and score of children with ADHD in mirror movement, compliance and temporal change of rotational size. Previous research on functional magnetic resonance imaging (MRI) reported activation of the ipsilateral cerebellum in typically developing adults during performing pronation and supination . MoreoveIn this study, we indicated that the pronation and supination in TD children and children with ADHD was only associated with age-appropriate development. We could not demonstrate whether our proposed method using acceleration and angular velocity sensors alone was useful for distinguishing children with ADHD from TD. We surmise that the pronation and supination may be associated not only with age but also differences of severity of ADHD or subtypes of ADHD: combined-type, inattentive-type, and hyperactive-impulsive-type. In this study, the number of children with ADHD was not enough to compare the differences in pronation and supination by severity of ADHD symptoms or by subtypes of ADHD. We need to measure more participants to answer this question in the future. SNS evaluation provides information about neurological conditions concerning motor function, learning, and behavioral problems . HoweverIn this study, we quantified some characteristics of ADHD children. From a comparison between TD children and children with ADHD by age, the pronation and supination function in children with ADHD has a tendency to lag behind that of typically developing children by several years. In conclusion, our system can be applied to evaluate the neurodevelopmental delay of children with ADHD. In the future, we plan to look into relationships between the pronation and supination function and the symptoms of different ADHD sub-types: combined-type, inattentive-type, and hyperactive-impulsive-type.


Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory “wild-type” strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference−silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element.Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the  Neurospora crassa has at least three genome defense mechanisms that have limited the colonization of its genome by selfish elements  . To protelements . RIP is elements . The thielements  and lateelements , occurs sad-1, a putative RNA-dependent RNA polymerase , a long interspersed element−like retroelement found intact and active in the Adiopodumé strain, has been demonstrated to transpose (N. crassa reference genome derived exclusively from FGSC 2489 (OR74A)  that originated from Sly1-1. Here, we present evidence that Sly1-1 is an active DNA-type transposon that is recognized by meiotic silencing when unpaired during meiosis. Furthermore, we confirm that the meiotic silencing machinery is required for the production of masiRNAs emanating from Sly1-1. Thus we provide support for the role of meiotic silencing in genome defense through detection of meiotic small RNAs targeting a TE in an unpaired state.By comparing genomes of several laboratory strains, multiple loci in the reference 2489 from the Fungal Genetics Stock Center  were identified to be missing among individuals in this pedigree. One of the largest of these detected insertion/deletions is a TE we named Supporting Information, Table S1. All strains were originally obtained from the FGSC and are maintained in the senior author’s laboratories. Mycelia of strains used for DNA extraction were collected after growth in liquid Vogel’s medium N . Tissues from these regions were scraped from cellophane and flash-frozen in liquid nitrogen. All collected tissues were stored at −80° until further use.Neurospora strains used in this study are listed in medium N  at 25° fmedium N  covered g for 15 min at 4°, the aqueous phase was transferred to a clean tube, and 500 μL of isopropanol for each 1 mL of Trizol was added. Precipitated RNA formed a compacted pellet after centrifuging at 13,000g for 20 min at 4° and the supernatant was removed. The pellet was washed with 80% ethanol, vortexed, centrifuged at 7500g for 5 min, and allowed to air dry for 10 min. Total RNA solution was obtained after dissolving the pellet in diethylpyrocarbonate-treated water. Polyethylene glycol8000 and NaCl were added into total RNA with final concentration 5% polyethylene glycol and 0.5 M NaCl to differentially precipitated high-molecular-weight RNAs followed by sitting on ice for 2 hr. Low-molecular-weight RNAs were recovered from the supernatant by ethanol precipitation, resolved by diethylpyrocarbonate-treated water and quantified in a NanoDrop 2000c spectrophotometer . Approximately 10 μg of isolated low-molecular- RNAs were separated on a 15% denaturing polyacrylamide-urea gel in with a miRNA marker was used as molecular mass standard . RNA was transferred to a Hybond-NX membrane  in 0.5× TBE using Trans-Blot Electrophoretic Transfer Cell apparatus  at 14V overnight. Constitutively expressed 18S ribosomal RNA was used as a control to test for equal loading of RNA by staining membranes with ethidium bromide for visualization. Carbodiimide-mediated crosslinking for 2 hr at 60° was used to crosslink RNA to Hybond-NX membranes followed by baking at 80° for 1 hr  per 50−100 mg of tissue and vortexed thoroughly. To each 1 mL of Trizol, 200 μL of chloroform was added, vortexed for 15–30 sec, and incubated at room temperature for 5 min. Samples were centrifuged at 13,000Table S4). The polymerase chain reaction (PCR) products were ∼500 bp and arranged end to end. Each of the amplicons were verified by sequencing and mixed together as templates to make 32P-labeled DNA probes. Prehybridization and hybridization was performed in PerfectHyb Plus hybridization buffer , at 42° overnight. To remove unspecific background, the membrane was washed twice in 2× saline sodium citrate  and 0.1% sodium dodecyl sulfate (SDS) at 40° for 15 min, and once in 0.5× SSC  and 0.1% SDS at 40° for 15 min. Finally, the membranes were exposed to a phosphoimager screen and scanned after 24 hr on a Typhoon 941 phosphoimager. Results were analyzed by Image Quant TL (version 7.0) software.Twelve primer pairs were used to amplify the NCU09969 locus  and 3 μL of Proteinase K (QIAGEN) at 60° for 1−2 hr. After cooling to room temperature, 200 μL protein precipitation solution (QIAGEN) was added to each sample and the solution kept on ice for 10 min. Samples were centrifuged at 13,000XhoI, AflIII, Ncol, DraIII, and Styl were used separately to digest ∼20 μg of genomic DNA from various strains. Digested products were separated in 0.8% agarose gels, followed by blotting onto nylon membranes . Four PCR products were used as templates to synthesize four 32P-labeled DNA probes . Probes A, B, and C were hybridized to blots with XhoI-digested DNA. Probe D was hybridized to blots with AflIII-, Ncol-, and DraIII-digested DNA. Prehybridization and hybridization was performed in PerfectHyb Plus hybridization buffer  at 65° overnight. Membranes were washed one time at 65° in four solutions  and 0.1% SDS for 15 min, respectively, to minimize unspecific background. The membranes were exposed to a phosphoimager screen and scanned after 24 hr on a Typhoon 941 phosphorimager. Results were analyzed by Image Quant TL (version 7.0) software.The restriction enzymes N. crassa β-tubulin gene (NCU04054) was used as an internal control for qRT-PCR. Each reaction was in triplicate and performed in a Bio-Rad CFX 96 Real-Time PCR machine. Primer sequences are listed in Table S5. Data analysis was performed using CFX Manager Software v3.1 to calculate the fold change using delta-delta Ct values. A sample from vegetative growth was used as control sample to calculate the relative RNA levels.Equal amounts (∼2 μg) of DNase I  treated total RNAs were reverse transcribed with SuperScript II reverse transcriptase (Invitrogen) using random hexamers. The 10-μL qRT-PCR system was used, including ∼50 ng of cDNA, 10 μL of iQ SYBR Green Supermix , and 150 nM primers. The Total RNAs from samples of three time points  were extracted with an miRNeasy Mini kit (QIAGEN). Small RNA sequencing library are constructed by following the standard protocols of Illumina TruSeq Small RNA Sample Prep kit in the University of Utah Sequencing Core. Size from 145 to 160 bp small RNA with adaptors (118 bp) were isolated and sequenced on an Illumina Genome Analyzer IIx to generate 50-nt single end reads. Sequence reads from three time points  are deposited in the SRA database under project accession number SRP021051. Additional pilot sequencing of the 4d PF time point, which had been previously prepared in a similar fashion and sequenced on an Illumina Genome Analyzer at University of British Columbia Sequencing center serves as replicate for comparison, though with considerably lower sequencing coverage.The genomic sequencing library of strain FGSC 8820 was constructed by using Nextera Illumina DNA preparation kit with dual indexing primers and sequenced in the Genomic Core at the Institute of Integrative Genome Biology, University of California, Riverside, on an Illumina HiSeq2000 genome analyzer. The sequence coverage was approximately 80X, and the reads are deposited in the SRA database under project accession SRP021049.http://hannonlab.cshl.edu/fastx_toolkit/). Reads longer than 17 nt and smaller than 30 nt were mapped to the reference genome assembly (N. crassa version 12 – accession AABX03000000.3) by Bowtie v2.1.0 (http://broadinstitute.github.io/picard). Identification of reads aligning to unpaired regions was performed using BEDtools v2.17.0 (http://github.com/stajichlab/neurospora_MSUD). An annotation file of noncoding RNAs was created by aligning the known sequences for ribosomal RNA , small nucleolar RNAs  and postprocessed with Adobe Illustrator.Illumina sequence reads in FASTQ format were processed to remove low quality and artifactual reads, trim Illumina adapter sequences with the fastx_toolkit. . Chromatin immunoprecipitation sequencing of the NMF229 strain also was analyzed in the same fashion. Sequences were trimmed for quality with sickle (http://github.com/najoshi/sickle) requiring at least phred quality of 30 and length of 25 bp. Paired end data processing required both pairs were required to pass filtering and quality control or else the entire pair was discarded. Trimmed and filtered short read DNA sequences were aligned to the genome with BWA v0.7.9-r783 using ‘bwa aln –q 20,’ which was further processed to produce SAM and sorted BAM files with SAMtools and Picard. Multiple matches were allowed to support the presence of multiple Sly-1 loci in the genome. Visualization of the read depth, and presence or absence of reads in the Sly1-1 locus was performed in the Genome Browser and computed using BEDtools v2.17.0 . A multiple alignment was constructed of these homologs and the Ch. globosum and Co. immitis copies and a phylogenetic tree constructed with RAxML (v7.3.2) (The superfamily classification of  domains  to ident(v7.3.2)  using a (v7.3.2)  and iden(v7.3.2) . The disSly1-1 including its 3-kb flanking DNA sequences upstream and downstream, using NCBI-BLASTN  were identified by self-aligning putative regions of Sly1-1 that have undergone RIP was done by RIPCAL  by computing RIP indices in sliding windows across all chromosomes. The RIP index for each locus was evaluated visually as a track in the Stajich lab Genome Browser .Testing for copies of y RIPCAL  based onhttp://gb2.fungalgenomes.org for visualization.Genome sequence, annotation, and BAM files for aligned reads were loaded into the Generic Genome Browser  to identify genomic origins of small RNAs. Analysis of read coverage indicated that some genomic regions were highly enriched in small RNA production, including subtelomeric, centromeric, and many rRNA and tRNA regions .To identify small RNAs produced from unpaired regions during meiotic silencing . Most 20-nt long RNAs were identified to originate from ribosomal DNA loci . We noted that the abundance of reads mapping to tRNAs decreased dramatically from 41.5% in PP to 16.9% in 2d PF and then increased slightly to 23.5% in 4d PF. It has been shown that the microRNA-like milRNA-4 is derived from the precursor of tRNA in vegetative tissue (Table S2). This class also showed preference for a 5′ uridine . This is expected for small RNAs processed by Argonaute-like proteins , suggesting that the formation of double-stranded RNA (dsRNA) is required.We next classified reads based on their match to genomic features into eight pools of small RNAs, namely rRNA, tRNA, small nucleolar RNAs, milRNA . Similarity analysis using a curated set of repetitive elements found eight of the regions contained the repeated sequences or remnants of TEs (Table S2).Analysis of the genomes of FGSC 2489 and FGSC 8820 was performed to identify regions unique to FGSC 2489 that could potentially form unpaired loops and trigger meiotic silencing. Previous work has shown that unpaired regions larger than 700 bp are needed to efficiently trigger meiotic silencing (Table S2). This region, located on linkage group (LG) VI , had a high abundance of small RNAs and contained two hypothetical genes, NCU09968 and NCU09969. We noticed that the read coverage of genomic sequencing of this locus in FGSC 2489 was greater than the flanking regions, which indicated that multiple copies of this region were present in the reference genome  while in Coccidioides immitis only the Sly transposase could be identified as gene (CIMG_13536).The original locus on LG VI contains a complete copy of GG_09452 . PhylogeGG_09452 , based oGG_09452  . The comparison between strains with no Sly1-1 and FGSC 2489 helped define the precise boundary of the transposon and the locations of the TIRs and TSDs and we observe that small RNA read abundance drops off sharply outside of the delineated locus . Only strains in the FGSC 2489 lineage . Our survey of all available re-sequencing data of N. crassa strains  (Sordaria macrospora (Sly1-1 is largely absent from the tested strains (data not shown); inspection of unreleased genome data from the pedigree of FGSC 2489 showed presence of Sly1-1 only in FGSC4200, FGSC9718, and FGSC987 . The absence of Sly1-1 in many of these strains suggests that it arrived or was activated only recently in N. crassa.To survey the presence of  strains , N. tetrSly1-1 sequence to identify the additional copy inferred by the second band in the Southern blots. A partial copy, which we named Sly1-2, is present in assembly 12 within centromeric DNA on LG II . In FGSC 2489 we found no evidence for RIP in the two Sly copies. The second copy may represent evidence of recent transposition. However, confidence in the assembly of the genome in this region is not high as repetitive regions are a hallmark of centromeric DNA and difficult to assemble. Because probes for both ends of Sly1-1 in the Southern blot showed evidence for two copies it is likely Sly1-2 is intact, just not assembled correctly . We also detected two additional loci that contained nearly identical TIR pairs, located on LG V and LG I; these were named Sly1-3 and Sly1-4, respectively . Sly1-3 and Sly1-4 are both absent in the genome of FGSC 8820. Sly1-2 is only about 3614 bp long, 100% identical to Sly1-1, has the same TSD as Sly1-1 adjacent to the sequence of TIR1 and contains a putative gene, NCU16528. The 3′ end of Sly1-2 is not completely assembled and stretches across two contigs. It is possible that Sly1-2 is larger in size and that its TIR2 is located in an unsequenced region. The unequal read coverage observed for the two loci in Sly1-1  while only one NCU09969 sequence is in the reference assembly at Sly1-1 which results in higher computed read coverage. Sly1-3 and Sly1-4 are very similar to each other: BLASTN showed query coverage ∼95% with identity ∼85% , both are ∼12 kb long, contain no identified open reading frames, and have nearly the same TSDs adjacent to TIRs. RIPCAL . The translated search of Sly1-3 and Sly1-4 also reveal similarity to both NCU09968  and NCU09969 , respectively. Summarizing all of these results, we conclude that Sly1-1 is a recently active TE, with at least two inactivated copies that underwent RIP (Sly1-3 and Sly1-4), but a partial copy which is identical in sequence and thus not mutated by RIP, Sly1-2, that may be active. Detection of similar but mutated copies of both genes, NCU09968 and sly1-1, in Sly1-3 and Sly1-4 suggests that the entire Sly1-1 locus transposed and not just the transposase, sly1-1.BLASTX searches with Sly1-1 as a consequence of meiotic silencing, we first determined when the sly1-1 transposase gene (NCU09969) was expressed. Analysis of published transcriptome data  values for sly1-1 from RNA-seq ranged from 6 to 10  locus in an unpaired and MSUD inducing cross . The size and expression pattern of Sly1-1 derived small RNAs were also confirmed by small RNA northern blots , to FGSC 2489. RNA collected at 2d, 4d, and 6d PF was tested by northern analyses. No small RNA from the Sly1-1 locus was detected under these conditions  to test for aberrant RNA production. Quantitative PCR showed that transcripts originating from downstream regions of the sly1-1 locus were highly induced from 2d PF to 6d PF . This observation suggested that RNA is required to initiate and maintain the production of aberrant and, presumably, small RNAs. In the cross between ΔSad-1 and FGSC 2489, transcripts accumulated at a high level at 2d PF, but decreased dramatically at 4d PF and 6d PF . This decrease in transcript levels indicated that the lack of SAD-1 did not block the production of aberrant, or any, RNA initially, but may have stalled dsRNA production, which resulted in the suppression of the production of small RNAs. The exact mechanisms underlying this surprising finding will need to be addressed by future experiments.We also tested whether the production of unprocessed N. crassa, a genome thought to be lacking active transposons  provided a better understanding of the origins and timing of acquisition of this newly described selfish genetic element. Our study also provides evidence for the original hypothesis that meiotic silencing targets unpaired DNA created by a transposon insertion and is an effective genome defense mechanism. The unpaired region triggers the production of masiRNA during sexual development, approximately 4−6 d after fertilization, when karyogamy is expected to occur.In summary, our study provides evidence for a novel and apparently intact TE in the widely used laboratory wild-type strain of