This is the third in a series of four cross-cultural empirical studies designed to develop International Classification of Functioning, Disability and Health  Core Sets for Attention-Deficit Hyperactivity Disorder (ADHD). To explore the perspectives of individuals diagnosed with ADHD, self-advocates, immediate family members and professional caregivers on relevant areas of impairment and functional abilities typical for ADHD across the lifespan as operationalized by the ICF(-CY). A qualitative study using focus group discussions or semi-structured interviews of 76 participants, divided into 16 stakeholder groups. Participants from five countries  were included. A deductive qualitative content analysis was conducted to extract meaningful functioning and disability concepts from verbatim material. Extracted concepts were then linked to ICF(-CY) categories by independent researchers using a standardized linking procedure. In total, 82 ICF(-CY) categories were identified, of which 32 were related to activities and participation, 25 to environmental factors, 23 to body functions and 2 to body structures. Participants also provided opinions on experienced positive sides to ADHD. A high level of energy and drive, creativity, hyper-focus, agreeableness, empathy, and willingness to assist others were the most consistently reported strengths associated with ADHD. Stakeholder perspectives highlighted the need to appraise ADHD in a broader context, extending beyond diagnostic criteria into many areas of ability and disability as well as environmental facilitators and barriers. This qualitative study, along with three other studies , will provide the scientific basis to define ICF(-CY) Core Sets for ADHD, from which assessment tools can be derived for use in clinical and research setting, as well as in health care administration.The online version of this article (doi:10.1007/s00787-017-0983-1) contains supplementary material, which is available to authorized users. Attention-Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder, affecting 3–7% of children and adults worldwide –4. BesidLevel 1 chapter: d5 self-careLevel 2 category: d570 looking after one’s healthLevel 3 category: d5702 maintaining one’s healthLevel 4 category: d57022 avoiding risks of abuse of drugs or alcoholIn 2001, the World Health Organization (WHO) released the International Classification of Functioning, Disability and Health (ICF) with the aim to provide a comprehensive and universal framework to describe different aspects of functioning and disability for all health-related conditions . In 2007Personal factors, such as gender, race, educational level, coping styles, are also deemed highly important and are included in the framework of the ICF(-CY). However, given their significant social and cultural variability, they have not been classified in the ICF(-CY).k = 531; Body structures, k = 329; Activities and participation, k = 552; and Environmental factors, k = 273). They serve to fine-map functioning and disability in all health conditions in research, clinical and healthcare administration settings for diagnostic, treatment, documentation and reimbursement purposes [The ICF(-CY), which includes all ICF categories plus additional ones for youth, consists of 1685 categories  categories. To facilitate comparison with our other ADHD Core Set preparatory studies , 35, an The study was approved by the regional ethical review board in Stockholm and by local ethics review boards in participating countries. Written and verbal informed consent was obtained from each participant and/or parent or legal guardian prior to participation. A qualitative methodology, combining focus group discussions and individual semi-structured interviews, was used for data collection. To achieve a broad representation, 76 participants were divided into 16 groups  having a well-documented primary clinical diagnosis of ADHD combined presentation or an ADHD subtype presentation (predominantly inattentive or hyperactive-impulsive) according to the diagnostic criteria of the ICD-10, DSM-IV/-TR or DSM-5 and/or receiving treatment for ADHD, (2) being an immediate family member, professional caregiver, or other closely involved in the everyday life of an individual fulfilling criteria (1). Participants were excluded from the study if they were younger than 7 years of age or could not communicate in the language of the country they resided in. Recruitment of participants was made via clinical research teams in each country, and via invitations in collaboration with local and national interest organizations for ADHD. In 10 stakeholder groups, data were collected through focus group discussions and the rest by employing individual-semi structured interviews. The contributions of stakeholder groups in respective countries were made by members of the project Steering Committee (see acknowledgement), a group of ADHD experts from all WHO-regions, who provide support and guidance on the project.For the group discussions and individual interviews, seven general items covering all WHO ICF(-CY) components (6 items) as well as one question on strengths associated with ADHD were included in the interview guide (see Online Appendix 1). The six items that covered all WHO ICF(-CY) components were predetermined, following the study protocol developed by the WHO and ICF Research Branch . To clarResearchers carefully examined the verbatim transcripts from the focus group discussions and individual semi-structured interviews. A deductive qualitative content analysis  was perfhttp://www.icf-research-branch.org). Linking results were compared and consensus discussions were used to resolve disagreements. In situations where consensus could not be reached, the coordinator at ICF Research Branch (MS) was available to make the final decision. However, this option was never used, as all disagreements were resolved by discussion. To investigate possible differences in inter-rater agreement between the two methods used for data collection (group discussions versus semi-structured interviews), Cohen’s Kappa was calculated separately. Kappa coefficients for the second level ICF(-CY) categories in the focus group discussions and semi-structured interviews were, respectively, ĸ = 0.68 (SE = 0.01) with a confidence interval of ĸ = 0.66–0.70, and ĸ = 0.70 (SE = 0.02) with a confidence interval of ĸ = 0.67–0.73. These indicate substantial agreement, irrespective of data collection method applied.To ensure the consistency of linking results for each focus group and semi-structured interview, both the identification of meaningful concepts and linking of ICF(-CY) categories were conducted by two independent researchers. To capture different cultural expressions in the participants’ answers, at least one independent researcher was included from the countries that were involved in this study . In total, seven independent researchers  were involved in the linkage. To prepare for the linking of actual data from the focus groups and individual interviews, the researchers received linking exercises that were provided by the ICF Research Branch  codes. To examine the consistency of quoted ICF(-CY) categories, orienting frequency analyses were conducted on the transcriptions across the different stakeholder groups. In this study, ICF-(CY) categories are presented at the second level. If a concept is linked to a third- or fourth-level ICF(-CY) category, the corresponding second-level category is reported. To avoid favouring participants that repeatedly expressed similar statements or were prompted by other participants’ responses, an ICF(-CY) category was only counted once for each stakeholder group that involved focus group discussion (max. 10) or individual semi-structured interviews (max. 6). Even though the possibility of prompting did not exist for those stakeholders who took part in individual semi-structured interviews, the same rule of counting was applied here to avoid favouring the responses of interviewed participants. Consistent with previous preparatory ICF Core Sets qualitative studies , 27, to n = 2) or late decline to participate in the study (n = 1). In addition, three children with ADHD were initially included in the study, but could not complete the focus group discussion due to restlessness. Table n = 17) along with the inattentive presentation (n = 12), followed by the hyperactive-impulsive presentation (n = 8) and unspecified ADHD (n = 2). Two participants chose to not respond to the question. Most immediate family members stated that their relative was diagnosed with the combined presentation (n = 12), followed by an inattentive presentation (n = 5). Only one family member indicated that their relative with ADHD was diagnosed with the predominantly hyperactive/impulsive presentation. Members of the interest organization mentioned they were in contact with individuals from the entire spectrum of ADHD (n = 4). Most commonly reported treatment methods by those diagnosed with ADHD were pharmacotherapy (n = 16), followed by pharmacotherapy and psychosocial treatment combined (n = 12). Two participants with ADHD received psychosocial treatment only, while eight did not report any current treatment for ADHD. The remaining three participants with ADHD did not respond to the question. Participating individuals diagnosed with ADHD lived with their parents (n = 3), independently (n = 3), with a partner (n = 3), or in communal settings (n = 3). The rest did not respond to the question (n = 2). Thirteen diagnosed individuals with ADHD reported to have university or college studies as their highest level of education, whereas twenty-six indicated high school or primary school studies as their highest level of education. Only one participant came from another level of education, namely vocational education, and one did not respond to the question. Individuals with ADHD were students (n = 25), full-time employed (n = 6), part-time employed (n = 2), self-employed (n = 2), engaged in volunteer work (n = 2), or held different forms of work and employment (n = 2), while one was on sick leave. Nine of those diagnosed with ADHD reported having at least one additional diagnosis.Of the 82 individuals eligible for study participation, 76 completed the group discussions or individual interviews. Attrition in 6 participants was due to not showing up for group discussions  categories, 243 personal factors , 152 non-definable codes , 120 not covered codes , and 17 health condition codes . Meaningful concepts that were linked to third or fourth level ICF(-CY) categories were aggregated to second-level categories. Different meaningful concepts that expressed similar functions were linked to the same ICF(-CY) category. For example, difficulties in relating with peers and maintaining friendships are two separate meaningful concepts, but they were linked to the same aggregated second-level ICF(-CY) category, namely d750 informal social relationships. When performing data saturation , only onk = 32), environmental factors (k = 25), body functions (k = 23) and body structures (k = 2). Table k = 6), d1 learning and applying knowledge , d2 general demands and tasks , d7 interpersonal relationships and interactions , d6 domestic life , d4 , d8 major life areas , d3 communication  and d9 community, social and civic life .Categories were found in each of the four ICF-(CY) components: activities and participation k = 3, environk = 8), e3 support and relationships , e1 products and technology , e5 Services, systems and policies  and e2 natural environment and human-made changes in environment .Second-level categories identified in the environmental factors component and their consistency across stakeholder groups are presented in Table k = 16). The other categories were identified in chapters b4 functions of the cardiovascular, haematological, immunological and respiratory systems , b5 functions of the digestive, metabolic and endocrine systems , b7 neuromusculoskeletal- and movement-related functions  and b2 sensory functions and pain .Table n = 5) and s7 structures related to movement   indicated positive sides to ADHD and named one or more strengths related to the condition. Only 7 (9%) did not report any positive side, and 15 (20%) felt unable to respond to the question. Out of the 15 participants who were unable to respond to the question, 13 (87%) of them were individuals diagnosed with ADHD, mostly children . Of those who reported no positive sides to ADHD, the large majority were immediate family members . Strengths reported included b130 energy and drive functions, which suggested to make it easier to engage in physical exercises  and to achieve personal goals and face general demands and challenges in life . Other examples included creativity, enabling to “think outside of the box”. Furthermore, d161 directing attention (hyper-focus) was mentioned as strength, provided that the activity or topic was a core interest of the individual. Specific positive attributes in b126 temperament and personality functions (i.e. agreeableness and willingness to assist others) were also recurrently mentioned. Table The majority of the participants  Core Sets for ADHD, the current international qualitative study aimed to investigate the experiences and perspectives of individuals with ADHD, self-advocates, immediate family members and professional caregivers on disability and abilities pertinent to ADHD, as well as facilitators and barriers to functioning. Categories were identified in all four ICF(-CY) components, mainly from activities and participation, but also several environmental factors and body functions. Very few body structures were considered to be relevant. The activities and participation component and environmental factors were described comprehensively, as evidenced by the fact that categories were covered in all nine, respectively, five chapters. In the body functions component, many different aspects of mental functions were considered to be important. Additionally, our study identified evidence of strengths associated with ADHD, such as high level of energy, creativity, hyper-focus, agreeableness, and willingness to assist others.The large number of ICF-(CY) categories identified across all of the components supports the notion that ADHD impacts on broad areas of body functioning and everyday life adaptive skills. Nearly 70% of all body functions categories covered in this study were from the b1 mental functions chapter, which is consistent with ADHD being conceptualized as neurodevelopmental and behavioural disorder. A total of 16 different mental functions were identified in this study, which demonstrates that cognitive functions are deemed crucial in ADHD. Our study also revealed physical alterations (e.g. body coordination) and sensory issues (e.g. sensation of pain) to be related to ADHD. The impact of ADHD on everyday life was described comprehensively. Consistent with previous studies on ADHD and social functioning , 36, thiContrary to our other preparatory studies, published in previous issues of this journal , 35, thiThis study is amongst the first to explore specific strengths in ADHD from an international and first hand perspective. Overall, the opinions stated were rather broad and not straightforward. In addition, several participants felt unable to mention any positive aspects related to ADHD, even when explicitly prompted. The most recurring themes about positive sides were creativity, high energy level, hyper-focus and sympathetic personality traits such as agreeableness and empathy. Still, there is currently little or no empirical support for such strengths outside of this study , 15. HowThe current study presents with some methodological challenges. The generalizability of the consistency of recurring ICF(-CY) categories across groups might be questioned, as the analyses were based on uneven sample sizes and compositions, i.e. half of the stakeholder groups came from Sweden and not all were equally represented. However, these orienting frequency analyses only reflect the consistency of ICF(-CY) categories across groups and were mainly conducted to facilitate comparisons with the other two previous preparatory studies , 35. TheDespite the limitations, this study managed to identify a wide range of functional abilities and disabilities in individuals with ADHD by involving multiple stakeholders and WHO-regions. Compared to previous ICF qualitative studies –29, thisSupplementary material 1 (DOCX 34 kb)Below is the link to the electronic supplementary material.


Recent research has shown high rates of comorbidity between attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) and difficulties regarding differential diagnosis. Unlike those in Western countries, the Japanese ADHD prevalence rate is lower relative to that of ASD. This inconsistency could have occurred because of cultural diversities among professionals such as physicians. However, little is known about attitudes toward ADHD and ASD in non-Western cultural contexts. We conducted two experiments to identify biases in ASD and ADHD assessment. In Study 1, we examined attitudes toward these disorders in medical doctors and mental health professionals, using a web-based questionnaire. In Study 2, medical doctors and clinical psychologists assessed four fictional cases based on criteria for ADHD, ASD, oppositional defiant disorder, and disinhibited social engagement disorder (DSED). Diagnosis of ASD was considered more difficult relative to that of ADHD. Most participants assessed the fictional DSED case as ASD, rather than DSED or ADHD. The results provide evidence that Japanese professionals are more likely to attribute children’s behavioral problems to ASD, relative to other disorders. Therefore, Japanese therapists could be more sensitive to and likely to diagnose ASD, relative to therapists in other countries. These findings suggest that cultural biases could influence clinicians’ diagnosis of ADHD and ASD. The behavioral, mood, and biological characteristics of mental disorders often overlap and occur comorbidly. Attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are the most difficult developmental disorders to distinguish from each other . ADHD isAlthough the main characteristics of ADHD and ASD differ, the two disorders overlap partially. For example, high rates of ADHD and ASD comorbidity have been reported , and indInterestingly, the prevalence of ADHD and ASD provided in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition DSM-5;  are incoStudies examining developmental disorders have been conducted mainly in Western cultures . HoweverI do not know the term, 2 = I have heard the term, 3 = I know something about the term, and 4 = I know the term well). According to the results, the proportion of people who were familiar with ADHD was higher than 20% (including I know something about the term and I know the term well), while over 60% of people were familiar with ASD . The most general sources of information included newspapers and television, indicating that there were numerous opportunities to obtain information concerning ASD in everyday life. Furthermore, Asian people characteristically attach high value to non-verbal information .Mage] = 43.03 ± 13.89 years). The participants did not receive compensation.We used Qualtrics.com to conduct a web survey. Written requests for responses were sent to 516 specialized agencies in the developmental support field in Japan . Either 10 or 11 organizations were randomly selected from each prefecture. The agencies were informed of the purpose of the study, voluntary nature of participation, and anonymity of the data collected. Therefore, each agency informed its constituents voluntarily. Participants were provided with similar explanations. The study was conducted from the beginning of March to the end of April 2014. Thirty-four adults participated in Study 1 , the numbers of diagnoses, and knowledge accuracy. Terms were based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision ; “even though ADHD and ASD are different disorders, it is not necessary to distinguish between them diagnostically” (differentiation unnecessary); “I believe that ADHD and ASD are the same disorder” (same); and “I do not consider ADHD and ASD disorders” (not disorders). The “differentiation unnecessary” and “same” statements were slightly similar. We assumed that participants who selected the former believed that ADHD and ASD were two distinct disorders, while those who chose the latter regarded ADHD and ASD as a single disorder.Participants chose the statement that described their own understanding most accurately from the following four statements regarding attitudes toward the relationship between ADHD and ASD, and provided a reason for the choice (we explained that there were no incorrect answers): “I consider ADHD and ASD different disorders” , which were adapted from those used by Participants reported the numbers of clients for whom they had provided treatment for each of the following diagnoses: ADHD, ASD, and comorbid ADHD and ASD (ADHD + ASD).All study participants provided informed consent; ethical approval was not required for this study in accordance with the national and institutional guidelines.∗Power 3.1  = 12.82 ± 10.20 years; mean number of correct responses to knowledge questions (Mcorr) = 14.29 ± 1.57. The participants were divided into two groups based on the following two criteria: whether they possessed the credentials required to diagnose patients, and whether they were involved with children in their professional work. Ten participants were medical doctors with the credentials required to diagnose patients , and 24 were other professionals who provided developmental support and did not possess the credentials necessary to diagnose patients .Participants’ characteristics were as follows: mean number of years of experience (t(32) = 2.8, p = 0.010, d = 1.04, as medical doctors’ mean age was higher than that of other professionals; however, there were no significant differences in years of experience, t(31) = 0.32, p = 0.80, d = 0.09, or numbers of correct responses to knowledge questions, t(12) = 0.96, p = 0.40, d = 0.45, between groups. Because the study included so few participants, and the accuracy of participants’ knowledge did not differ significantly between the two groups, we analyzed the responses from the two groups together.The main effect of group was significant with respect to age, 2(3) = 23.58, p < 0.001, w = 0.872, power = 0.990; different = 61%, differentiation unnecessary = 23%, same = 10%, and not disorders = 6%. The different statement was chosen more frequently than same and not disorders ; however, the difference between different and differentiation unnecessary only approached significance (p = 0.058). The frequencies for the other three statements did not differ significantly, ps > 0.10. In addition, the numbers of participants who did and did not select the different statement  did not differ significantly and the effect size was small . The results regarding the free descriptions are shown in Table 1.Three participants were excluded from the analysis because their reasons for their choices were unclear. The frequencies for the four statements differed significantly and we found a large effect size, χResponses that had unclear meanings or did not conform to the questions were excluded. Therefore, data from four participants were excluded from the analysis of psychological similarities and three participants were excluded from the analysis of psychological differences.Table 2 and Supplementary Table The analysis of psychological similarities included data for 30 participants. The summaries regarding psychological similarities concerned “overt characteristics” (87.3%), “secondary issues” (7.3%), and “intervention” . The “overt characteristics,” “secondary issues,” and “intervention” summaries included the responses observed for similarities, and “causes” concerned the causes of overt characteristics. Four responses clearly indicated that ADHD involved more serious issues, relative to those involved in ASD (represented by the number of responses indicating that the issues described in the items were most serious in ASD), and 14 suggested that ASD involved more serious issues than those involved in ADHD . A significantly higher number of responses suggested that serious difficulties were more numerous in ASD  than in ADHD and the effect size was large .The analysis of psychological differences included data for 31 participants. The summaries regarding psychological differences concerned “overt characteristics” (63.5%), “causes” (14.6%), “interventions” (7.3%), “secondary issues” (4.9%), and “other” ; “brain issues and genetic factors,” which concerned ADHD- and ASD-related problems regarding brain activity and genetic factors (12.9%); “genetic factors,” which concerned the ease that both ADHD and ASD are inherited (9.7%); “other,” which included hypersensitivity (9.7%); and “nothing,” which indicated that participants saw no similarities or did not know (35.5%).Responses concerning biological differences were classified into three categories: “brain region with problem,” “other,” and “nothing.” The “brain region with problem” category included problems involving the prefrontal region in ADHD and multiple regions in ASD; however, 12 out of 14 responses did not indicate detailed regional differences . The “other” category included the belief that ADHD symptoms would reduce with development, but ASD symptoms would not, or the belief that ADHD was likely to involve comorbidity, but ASD was not (9.7%). “Nothing” indicated that participants saw no differences or did not know (45.2%).2(1) = 2.613, p = 0.106, w = 0.290, power = 0.365; differences: χ2(1) = 0.290, p = 0 .590, w = 0.097, power = 0.084). We performed chi-square tests to analyze the relationships between the frequency with which different was chosen for conceptual understanding and recognition of the presence of biological similarities and differences (Table 4). Recognition of the relationship between ADHD and ASD was not significantly associated with recognition of biological similarities, χ2(1) = 0.056, p = 0.813, w = 0.119, power = 0.098, nor with biological differences, χ2(1) = 0.276, p = 0.599, w = 0.170, power = 0.150. The frequency of participants who believed in certain biological differences did not differ between participants who did and did not choose the different statement.Participants were divided into two groups unlike psychological similarities and differences: those who believed in certain biological factors and those who did not. The number of participants who saw similarities and differences between the two disorders in the group that believed in certain biological factors did not differ significantly relative to that observed in the group that did not believe in certain biological factors and the effect size was small, especially regarding biological difference [similarities: χF = 22.78, p < 0.001, ηp2 = 0.416. The diagnosis rate for ASD was significantly higher than that for ADHD  and ADHD + ASD . There was no significant difference between diagnosis rates for ADHD and ADHD + ASD .The mean diagnosis rates were as follows: ADHD = 28.41 ± 13.45, ASD = 49.00 ± 13.66, and ADHD + ASD = 23.52 ± 12.97. The results of the one-way ANOVA showed that the main effect of diagnosis was significant, different statement) did not differ significantly. Further, the frequency of participants who believed in biological similarities and differences did not differ markedly from the participants who did not believe in biological factors, unlike psychological factors. The results are discussed in detail below.The aim of the study was to determine whether and how bias in the understanding of ADHD and ASD existed in the Japanese population. The results showed that participants focused more closely on difficulties concerning ASD, relative to those concerning ADHD, in their free descriptions regarding psychological differences. Further, the diagnosis rate for ASD was significantly higher than those for ADHD and ADHD + ASD. This finding differs from previous studies, in which the prevalence rate for ADHD was higher than that of ASD . HoweverThe results showed that participants focused more strongly on the overt characteristics of ASD  relative to symptoms such as ADHD-related inattention, impulsivity, and hyperactivity, when considering the psychological similarities of ADHD and ASD. Furthermore, participants referred to ASD more frequently than ADHD in the free descriptions of the psychological differences between ADHD and ASD. These results are consistent with our hypothesis that participants would focus on non-verbal communication skills. Based on previous studies that showed individuals with ASD presented with ADHD-related impulsivity and inattention  and thosdifferent statement believed that ADHD- and ASD-related characteristics and/or interventions differed. However, some participants focused on the need for individualized support or believed that the disorders were on the same spectrum. It is possible that recognition of individualized treatment needs prevented participants from distinguishing between ADHD and ASD, and this, in addition to the above-mentioned biases, led to weak diagnostic agreement. Considering the insufficient knowledge about biological factors, greater clarification and dissemination of information regarding the biological similarities, differences, and overlap between the disorders could be useful in improving differential diagnoses for ADHD and ASD and enhance evaluation tools designed to determine whether individuals have one or both disorders.The results also showed that participants’ recognition of the relationship between ADHD and ASD did not necessarily correspond to operational diagnostic criteria. Even though ADHD and ASD are similar in that they are both neurodevelopmental disorders, they are classified as separate disorders with distinct diagnostic criteria. Participants who chose the If Japanese people are biased, in that they find it easy to focus on ASD-related characteristics specifically and separate them from the multiple other possible symptoms, this could affect the accuracy of assessments and lead to weak diagnostic agreement . Therefore, we conducted a second experiment to determine whether participants exhibited sensitivity to ASD.Based on the results of Study 1, we assumed that the participants, who could affect the assessment of children’s development, were more sensitive to ASD symptoms than those of ADHD. However, the results of Study 1 did not explain how this bias affected assessment. If participants were sensitive to ASD-related symptoms in examining children’s behavioral or emotional characteristics, they could have regarded ADHD-related characteristics as signs of ASD. Moreover, they could have suspected that mental and behavioral symptoms that were similar to those observed in ADHD in the diagnostic criteria were related to ASD rather than ADHD.For example, the DSM-IV-TR  and DSM-These characteristics tend to be difficult to distinguish from those of ADHD. However, if there is bias in the tendency to attribute symptoms to ASD, these characteristics could be more likely to be confused with ASD than ADHD. In Study 2, we examined clinicians’ assessment tendencies using the original assessment task.We assumed that participants in Study 1 recognized the similarities and difficulties involved in differentiating ADHD from other mental problems; however, it was possible that participants were unaware of them, even though bias was observed for ADHD and ASD. Therefore, we confirmed subjective similarities and related difficulties in differentiation.We searched for three or four specialized agencies in the mental support field for children  per prefecture. All Societies of Certified Clinical Psychologists in Japan were included, because each prefecture contained a society. In the hospital selection process, we chose larger hospitals  because we assumed that they employed more medical doctors than private hospitals. In prefectures with numerous hospitals, we selected large hospitals randomly.Written requests for responses to a web survey, which was conducted via Qualtrics.com, were sent to 164 agencies. The agencies and their constituents received information regarding the study purpose, the voluntary nature of participation, and anonymity, as in Study 1. The study was conducted between October 2014 and April 2015.Mage = 39.23 ± 10.34 years, Mexp = 9.10 ± 6.94 years). Participants consisted of 12 pediatricians and psychiatrists  and 41 clinical psychologists .Fifty-three medical doctors who specialized in children’s mental health and clinical psychologists who supported children at elementary school, and who had not participated in Study 1, participated in Study 2  in the web-based research.Participants were asked to read four fictional cases  and diagnose the mental health problems that manifested as internal and/or external problems for each case. The instructions were as follows: “The following four questions are about fifth-year pupils’ behavior at school; the pupils are enrolled in a general education class at elementary school. If you suspect mental health or behavioral problems for each child, what do you suspect? If you feel that there is insufficient information available for assessment, please imagine what you suspect as the potential problem, using the information provided, and engage in subsequent information gathering and support provision. There are various possible responses to the four questions, and duplicate responses are permitted.” We used the phrase “mental health or behavioral problems” rather than “mental health problems” alone to provide participants with images of internal and/or external problems from the vignettes. Assumptions of comorbidity and multiple responses were permitted, and participants were asked to prioritize multiple responses.extremely dissimilar) to 7 (extremely similar). In the difficulty ratings, participants were asked to indicate the extent of the difficulty they experienced in distinguishing between ADHD and other problems , using a seven-point Likert scale ranging from 1 (very easy) to 7 (very difficult). In both tasks, participants chose “unknown” if they had not previously heard of a disorder.Participants rated the similarities and difficulties involved in distinguishing between ADHD and other mental health problems. In the similarity ratings, participants were asked to indicate the extent to which they felt ADHD was similar to certain other conditions , using a seven-point Likert scale ranging from 1 . Prior to the rating exercise, we asked participants to indicate whether they had ever heard of the eight above-mentioned disorders by selecting either “I have heard of it” or “I haven’t heard of it.”To confirm whether assessment standards were consistent among participants, we examined the diagnostic criteria (definitions) participants usually used to assess mental health or behavioral problems in children. Participants were asked to number the following criteria in ascending order according to usage: DSM-IV-TR, DSM-5, International Statistical Classification of Diseases, Tenth Revision (ICD-10), and other (describe explicitly).All study participants provided informed consent. Ethical approval was not required for the study.∗Power 3.1  were used to compare rating values among eight similarity characteristics and 11 difficulty characteristics.Table 5). Accuracy in the four tasks differed significantly, Cochran’s Q(3) = 71.45, p < 0.001. The results of McNemar’s chi-squared test showed that the accuracy of responses to questions regarding cases involving ASD and ADHD was higher than that for questions regarding cases involving ODD and DSED, ASD vs. ODD: χ2(1) = 29.03; ASD vs. DSED: χ2(1) = 26.69; ADHD vs. ODD: χ2(1) = 26.04, ADHD vs. DSED: χ2(1) = 25.29, all ps < 0.001. The accuracy of responses did not differ significantly between cases involving ASD and ADHD, χ2(1) = 0.44, p = 0.505, or ODD and DSED, χ2(1) < 0.001, p > 0.999.The correct response rates were 86.8, 92.5, 32.1, and 34.0% for the questions regarding cases involving ADHD, ASD, DSED, and ODD, respectively (p < 0.001) and ASD (p < 0.001) were significantly higher than numbers of incorrect responses. In addition, the number of incorrect responses to questions regarding cases involving ODD (p = 0.027) and DSED (p = 0.013) was significantly higher than the number of correct responses. Furthermore, the most common incorrect response to the question regarding the case involving DSED was “ASD” (37.7%).The numbers of correct responses to questions regarding cases involving ADHD  = 136.70, p < 0.001, η2 = 0.416, power > 0.999; difficulty: χ2(10) = 98.83, p < 0.001, η2 = 0.210, power = 0.999. Multiple comparisons of the Wilcoxon signed-rank test (Bonferroni method) indicated that the mean values for ASD, ODD, and DSED were significantly higher than those for anxiety disorder (ps < 0.05), depressive disorders (ps < 0.01), and intellectual disabilities (ps < 0.05), for both similarity and difficulty , 20 (37.74%), five (9.43%), and four , respectively.To determine whether medical doctors and school counselors who supported school-age children were more sensitive to ASD-related than ADHD-related characteristics, we administered a questionnaire involving four assessment tasks  and examined subjective rates of similarity and difficulty in distinguishing between ADHD and other mental health problems. In the assessment task, accuracy rates for cases involving ASD and ADHD were higher than those for cases involving ODD and DSED. Furthermore, the case involving DSED was assessed mainly as ASD, rather than DSED or ADHD. The results also showed that participants considered ASD, ODD, and DSED as similar to ADHD. Moreover, although the criteria used by participants daily could have influenced assessment results, almost all participants based their assessments on common operational diagnostic criteria.The numbers of correct responses to questions regarding cases involving ASD and ADHD were higher than the numbers of incorrect responses. Based on the results of Study 1, we hypothesized that ADHD would be confused with ASD; however, this hypothesis was not supported. This finding could have occurred because we used typical behavioral characteristics in the fictional cases, and participants were likely to diagnose ADHD, rather than ASD, when children presented with typical ADHD-related characteristics.The similarity and difficulty ratings indicated that DSED was considered similar to ADHD, and participants found it difficult to distinguish between these two disorders. This accords with the DSM-5 , which sAlthough clinicians showed elevated levels of accuracy in differentiating between ADHD and ASD, our original concern remained. In practice, children present with various characteristics including normal activity, inattention, depression, and strong preferences; from these, mental health professionals are usually required to detect abnormal traits. However, considering participants’ difficulties in diagnosing DSED, it is possible that they were biased toward ASD-related features in their assessment of children with not only hyperactivity, impulsivity, and inattention but also interpersonal problems.In addition, considering the high accuracy rates for ADHD and ASD assessments and low accuracy rates for ODD and DSED assessments, differences in familiarity with developmental and other psychiatric disorders could have influenced the assessment results. One probable reason for this finding is that participants could have been more familiar with and likely to diagnose developmental disorder relative to other disorders, as a few participants had not heard of “DSED,” “ODD,” or “conduct disorder.” When unsure, the participants might assess children’s behavior as ASD because they are more familiar with it.The results of the study showed inconsistencies and biases in understanding regarding ADHD and ASD in individuals who performed psychological assessments in the Japanese population. Almost half of the participants in Study 1 understood that ADHD and ASD are distinct disorders; however, the remaining participants did not. In addition, participants in Study 1 focused more strongly on ASD relative to ADHD. Moreover, based on the results of Study 2, mental health professionals could exhibit a tendency toward confusing DSED with ASD.The service climate, with respect to medicine, education, and welfare, in each country should be considered when discussing biases in assessment and diagnosis. To diagnose children with ASD is not considered to provide greater benefits than diagnosing other mental health disorders, including ADHD, in Japan, as children with any type of mental health disorder are almost equally eligible to receive welfare, medical, and educational services; mental disability certificates for the provision of financial aid; pharmacotherapy; and social skills training. However, despite this equality in the service climate, we conclude that the attitudes of medical doctors and other professionals could be biased toward ASD, as the participants’ focus on ASD-related characteristics was stronger than that on ADHD- and DSED-related characteristics in both experiments.The rating of child hyperactive-disruptive behaviors by mental health professionals differs per culture ; therefoSome studies of psychological assessment have noted the impact of cultural differences via internationalization. Main concerns included how children’s ethnic diversity affects mental health professionals and/or teachers’ decisions regarding participants’ behavior e.g., , how menRegarding bias in assessment, When therapists assess children’s psychiatric traits, they usually use multiple assessment methods and test batteries . When assessing ASD or ADHD, diagnostic schedules and questionnaires . Each agency was informed about the purpose of the research, voluntary nature of participation, and anonymity of the responses collected. The agencies could inform its constituents. Therefore, participants were recruited only if the agency confirmed that the study was appropriate for its constituents. Second, the same details were provided to the participants. Before participants answered the questionnaire, we set opportunities at different times for agencies or participants to choose whether they wanted to join the study. In addition, participants were allowed to quit answering the questionnaire anytime they wanted. Although our study has not been approved by the research ethics committee, we believe that the voluntary participation and effort of participants is significant. Therefore, our study complies with the ethical policy of your journal.MM, SK, and MN: Study conception or design, interpretation of study data, drafting the manuscript or revising it for critical intellectual content, final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. MM: acquisition and analysis.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.


Viperin is a multifunctional interferon-inducible broad-spectrum antiviral protein. Viperin belongs to the S-Adenosylmethionine (SAM) superfamily of enzymes known to catalyze a wide variety of radical-mediated reactions. However, the exact mechanism by which viperin exerts its functions is still unclear. Interestingly, for many RNA viruses viperin was shown to inhibit viral RNA accumulation by interacting with different viral non-structural proteins. Here, we show that viperin inhibits RNA synthesis by bacteriophage T7 polymerase in mammalian cells. This inhibition is specific and occurs at the RNA level. Viperin expression significantly reduced T7-mediated cytoplasmic RNA levels. The data showing that viperin inhibits the bacteriophage T7 polymerase supports the conservation of viperin’s antiviral activity between species. These results highlight the possibility that viperin might utilize a broader mechanism of inhibition. Accordingly, our results suggest a novel mechanism involving polymerase inhibition and provides a tractable system for future mechanistic studies of viperin. Viperin is an IFN stimulated antiviral protein that inhibits a broad spectrum of both DNA and RNA viruses3. Interestingly, viperin was found to inhibit different viruses at different steps of the viral life cycle suggesting that this protein has multiple functions. Viperin was found to protect the infected cells by affecting lipid raft formation and modulating cellular metabolism5.The interferon (IFN) response is one of the first lines of host innate defense against viral infections. Pattern recognition sensors in the infected host cell recognize the incoming pathogens and initiate signal transduction cascades that culminate in the host cell’s nucleus. These signaling cascades activate type I IFN production and the induction of interferon-stimulated genes (ISGs) aimed to prevent infection in an autocrine and paracrine manner7. These enzymes contain a consensus Cx3Cx2C motif responsible for binding iron-sulfur clusters to preform reductive cleavage of S-Adenosylmethionine (SAM) to generate a deoxyadenosyl radical and methionine8. The obtained radical is highly reactive and is known to mediate a variety of reactions9. No specific enzyme activity, however, has been yet assigned to viperin. The Fe-S binding motif, however, seems to be essential for its antiviral activity against some viruses11. Viperin also requires an additional protein, cytosolic iron-sulfur assembly component 1(CIAO1) crucial for the Fe/S cluster insertion and thus for its SAM activity and accordingly for its antiviral activity against the aforementioned viruses12. The broad range of viperin-affected viruses across taxonomic groups impedes the efforts to identify its specific antiviral effects. Interestingly, in many RNA viruses viperin was shown to inhibit viral RNA accumulation18. Moreover, viperin inhibited Hepatitis C, Dengue and West Nile virus replication from subgenomic replicons19. Subgenomic replicons contain only the viral non-structural proteins essential for viral replication and lack the viral structural proteins, further confirming that viperin inhibits viral replication. Mechanistically, viperin was shown to interact with non-structural proteins of hepatitis C virus and dengue virus that are essential for viral replication. These interactions are thought to be associated with its antiviral activity17. Here we describe a substantial inhibitory effect of viperin expression on bacteriophage T7 polymerase mediated transcription in mammalian cells. The fact that viperin inhibits transcription by a bacteriophage polymerase supports the conservation of viperin’s antiviral activity between species. Inhibition of the viral transcription might be a compelling possible mechanism for viperin’s mode of action. Several recent findings support such a hypothesis; Oxetanocin A, an antiviral nucleoside analogue produced in bacteria, was found to be biosynthesized by a radical SAM enzyme20. This suggests that SAM radical family enzymes can generate antiviral products. Furthermore, structural similarity studies of the active site of viperin, based on the crystal structure of murine viperin, suggest that the substrate may be a nucleoside triphosphate21.Viperin is a member of the “Radical SAM” family of enzymes22. T7 RNA polymerase functions in the cytoplasm and thus is the most widely used RNA polymerase in viral reverse genetic systems driving the initial transcription of RNA viruses replicating in this compartment24. Here we describe a substantial effect of viperin on bacteriophage T7 polymerase activity in mammalian cells. Overexpression of viperin strongly inhibited green fluorescent protein (GFP) expression from a T7 promoter (T7-GFP). Viperin did not inhibit RNA polymerase II dependent RNA synthesis from a Cytomegalovirus (CMV) promoter under similar conditions. This inhibition was specific and not at the level of translation. Mutagenesis of viperin’s functional domains inhibited this activity. Following T7-GFP transfection, 5′-bromouridine 5′-triphosphate (BrUTP) incorporation experiments showed significantly lower levels of RNA synthesis in the cytoplasm of viperin-expressing cells. Previous studies show that viperins are highly conserved and that fungi, bacteria, and archaebacteria express viperin-like enzymes21, indicating that viperin’s effect on T7 polymerase might be conserved. Taken together our results support the conservation of viperin’s antiviral activity between species and suggest a common mechanism of inhibition might be responsible of these activities.Bacteriophage T7 DNA-dependent RNA polymerase (T7 polymerase) is a well-characterized RNA polymerase. T7 polymerase is widely used as a model to study transcription and in a wide verity of molecular biology applications due to its robust activity and promoter specificity25. A plasmid containing Renilla luciferase was used as a control. Overexpression of viperin strongly inhibited the expression of T7-GFP. Interestingly, this inhibition was specific for expression from the T7 promoter and did not affect the expression of a cyan fluorescent protein (CFP)- yellow fluorescent protein (YFP) fusion from an RNA polymerase II-dependent CMV promoter  with increasing levels of viperin and tested its effect on GFP expression from a T7 or CMV promoters .While conducting a screen for host factors affecting a virus using a T7-polymerase based reverse genetics system, viperin was identified as a potent T7 polymerase inhibitor. In this screen, we expressed various ISGs from a bicistronic lentivirus also expressing Red fluorescent protein (RFP) as an infection controlter Fig. . Of noteter Fig. . A diffeter Fig. . This isers Fig. . A dose ers Fig. . To confers Fig. . Viperiners Fig. . Similar28. A radical SAM domain containing the Cx3Cx2C motif important for binding an iron–sulfur cluster responsible for its enzymatic activity7. The third domain, located at the highly-conserved C-terminal of the protein, was found to mediate interactions with host proteins essential for viperin’s enzymatic activity and antiviral functions18. To gain insight into the mechanism by which viperin inhibits RNA synthesis, we constructed a series of viperin mutants containing mutations in the above-mentioned functional domains. These mutants contained: Δ42 N a deletion of the N-terminal 42 amino acids (a.a.), Δ33C a deletion of 33 a.a. from the C-terminal and S1, containing a point mutation changing the first cysteine in the Cx3Cx2C motif into alanine  and to lipid dropletsets Fig. . Viperinets Fig. . In contion Fig. , indicat30. To test this possibility, global protein synthesis rate in the presence of viperin was tested using puromycin labeling31. Increasing amounts of viperin were transfected into HEK293T cells. After transfection (48 h), the cells were incubated with puromycin, after which they were washed, lysed and analyzed by Western blot using puromycin-specific antibodies. Fig. 32 , suggesting that viperin does not affect pre-synthesized RNA. A nuclear localized T7-polymerase was generated to examine if there is a direct interaction between viperin and T7 polymerase. In addition this construct was used to test if viperin-mediated inhibition is limited to a specific localization of the T7-polymerase. The nuclear T7-polymerase was generated by introduction of a nuclear localization signal (NLS) into the N-terminal of the T7-polymerase. The NLS-T7-polymerase expressing plasmid was transfected into BHK-21 cells together with T7-GFP and its intracellular localization was determined using a T7-polymerase specific antibody  incorporation into newly synthesized RNA experiments were performed. This method enabled us to visually distinguish between RNA synthesis in the nucleus and the cytoplasm. To establish the system, control or high multiplicity of infection (MOI) Vesicular stomatitis virus (VSV) infected BHK-21 cells were used. Following infection, the cells were transfected with BrUTP, incubated at 37 °C and then fixed. BrUTP containing RNA was detected using a bromouridine specific antibody. Actinomycin D (ActD), an inhibitor of DNA dependent RNA transcription, was used as a negative control36. As expected, control uninfected cells displayed only nuclear staining that was abolished by the addition of ActD . Furthermore, viperin-expressing cells show lower levels of BrUTP expression 1 h post BrUTP transfection. An effect within such a time frame is most likely on RNA synthesis rather than degradation.42. Thus, T7 polymerase is fully functional in mammalian cells and in can generate high levels of transcription in the cytoplasm of many cell types confirming its relevance. Additional support for its relevance comes from the finding that viperins are highly conserved and organisms from fungi to bacteria and archaebacteria also express viperin-like enzymes21. Moreover, human viperin was found to cause radical SAM- dependent elongation of E. Coli indicating that its substrate might be shared between bacteria and humans43.The question of the relevance of viperin’s activity against the T7-dependent expression to mammalian biology should be raised. The T7 polymerase system is a widely-used system to initiate cytoplasmic replication in reverse genetics systems of many viruses18. Viperin was shown to bind to viral non-structural proteins associated with viral replication17. The observed inhibition of the T7 polymerase might indicate a different mechanism since in this system RNA synthesis does not occur in membrane complexes or requires additional bacteriophage proteins. BrUTP experiments in viral systems were so far unsuccessful due to low efficiency of transfection in the infected cells. Nevertheless, this highlights the significance of our findings in providing a highly tractable system to study this activity of viperin. If viperin indeed inhibits RNA synthesis, it seems to use a mechanism that inhibits a rather broad range of polymerases; RNA-dependent RNA polymerases from different viruses and a bacteriophage DNA-dependent RNA polymerase. Thus, it is less likely that viperin directly binds these structurally different RNA polymerases. In addition, our immunoprecipitation experiments, which do not detect such an interaction, suggest otherwise. An in vitro assay would be useful in the identification of a direct effect of viperin on the polymerase. However, since viperin is a membrane-associated protein28, requires an additional protein partner for its activity11, might require addition of nucleoside precursors21 and needs to be purified under anaerobic conditions, establishment of such an in vitro assay will be extremely challenging. One possible option is that viperin inhibits a host cell factor essential for viral/cytoplasmic RNA synthesis (for examples of such factors see45). Alternatively, viperin might affect a general RNA synthesis resource that is more frequently utilized during high levels of RNA synthesis. For example, viperin might affect nucleoside synthesis or modify a nucleoside to create an antiviral nucleoside analogue. Support for the latter possibility may come from a recent finding that viperin’s substrate is a nucleoside triphosphate21. In addition, Oxetanocin A, a nucleoside analogue produced in bacteria with an antiviral activity is produced by a SAM radical family member20. Alternatively, a subset SAM radical enzymes were found to catalyze methylation reactions46. For example, RlmN and Cfr post-transcriptionally methylate ribosomal RNA nucleotides47. Since neither cap nor IRES-dependent translation is inhibited by viperin  supplemented with 10% (v/v) fetal calf serum, 1% (v/v) penicillin/streptomycin . The HEK293-T7 and BSR-T7 cells were grown in the presence of hygromycin  or G418  respectively. Lipofectamine 2000  or polyethyleneimine  were used for plasmid DNA transfections of subconfluent cells. Vesicular stomatitis virus (VSV) strain ts045 was propagated and tittered in Vero cells as describedpTRIP.CMV. IVSb.ISG.ires.TagRFP were a gift from Prof. Charlie Rice25 (Rockefeller university). The bicistronic plasmid expressing dsRed in a cap dependent manner and GFP from an IRES32 was a kind gift from Prof. C.G Proud (University of Dundee).T7-GFP and the T7 polymerase expressing plasmids were a kind gift from Prof. Shmuel Rozenblatt (Tel-Aviv University). The c-Myc NLS signal was added to the N-terminus of the T7 polymerase by PCR using the following primers: FW: CCAAGCGAGTTAAATTAGACAATACCATAAACATTGCTAAGAACG. RV: CAGCAGGCATGGGTACCCGGTGTCTTCTATGGAGGTC. The original plasmid was eliminated using the DpnI enzyme, the PCR product containing the entire original plasmid plus the NLS signal was gel-purified, ligated and transformed into bacteria. pEGFP-C1 , was used to express GFP from a CMV promoter. The T7-firefly luciferase and the ß-galactosidase expressing plasmids were from Promega . HEK293-T7 a pool of T7 polymerase expressing HEK293 cells was a gift from Prof. Eran Bacharach (Tel-Aviv University). Calnexin-GFP was a gift from Prof. Rina Arbesfeld (Tel-Aviv University). For the T7-GFP and T7-luciferase quantification experiments human viperin was cloned into pCAGGS plasmid using Gibson assembly. Briefly, viperin was amplified from a viperin-F.LAG plasmid  with the primers primers Fw 5′-GAATTCGAGCTCATCGATGCATGGTACCATGTGGGTGCTTACACCTGCTG-3′ for Fw and Rev 5′-CATAATTTTTGGCAGAGGGAAAAAGATCTCTACCAATCCAGCTTCAGAT C-3′ and ligated into pCAGGS digested with KpnI and BglII. The viperin mutants were similarlly cloned into pCAGGS. The primers used for Δ42 N were Fw 5′-GAATTCGAGCTCATCGATGCATGGTACCATGCTAGCTACCAAGAGGAG-3′. For Δ33 C Rev 5′-ATAATTTTTGGCAGAGGGAAAAAGATCTCTATTCATCCAGAATAAGGTAG-3′. For the S1 mutant two overlapping mutation-containing viperin fragments used for Gibson assembly into the afore-mentioned sites. The primer used for the first fragment: Fw 5′-GAATTCGAGCTCATCGATGCATGGTACCATGTGGGTGCTTACACCTGCTG-3′, Rev 5′-GCGAAGCCGGCTTTGTAGTTGGCCTGGCGAGTGAAGTGATAGTTG-3′ and for fragment 2: Fw 5′-GCCAACTACAAAGCCGGCTTCGCTTTCCACACAGCCAAAACATC-3′ Rev 5′-CATAATTTTTGGCAGAGGGAAAAAGATCTCTACCAATCCAGCTTCAGATC-3′. The CFP-YFP fusion expression plasmid was a gift from Prof. Koret Hirschberg (Tel-Aviv University). ISG expressing lentiviruses 49 and normalized to the loading control. Values are presented as percent of the luciferase control.Cell were lysed in lysis buffer containing 300 mM NaCl, 100 mM Tris–HCl pH 8, 0.2 mM EDTA, 10% Glycerol, supplemented with a protease inhibitor cocktail . The lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in blocking solution containing 5% non-fat milk  in phosphate buffer saline (PBS) followed by incubation with the appropriate primary antibody. Primary antibodies used were rabbit polyclonal anti-RFP antibody  and a rabbit anti-GFP polyclonal antibody . Anti-Viperin Antibody, clone MaP.VIP was from Millipore  and anti-T7 polymerase from Creative biolabs, Shirley, NY. Monoclonal mouse antibodies against β actin and tubulin were from Sigma. The blots were incubated with IR-Dye conjugated secondary antibodies  and visualized using a LI-COR infrared imager (Odyssey). Band intensity was quantified using imageJHEK293-T7 cells were plated to 15-cm plate and transfected the next day with 15 μg of viperin or a control plasmid. The cells were harvested 48 hours post-transfection and lysed with the M2 lysis buffer (50 mM Tris-base (pH 7.4), 50 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5 mM EDTA, 0.5 mM EGTA) supplemented with a protease inhibitor cocktail (Sigma). Cell lysates were incubated for 3 h with anti-viperin antibody conjugated to protein A/G agarose beads  at 4 °C. Beads were collected by slow centrifugation, washed four times with lysis buffer and analyzed by SDS–polyacrylamide gel electrophoresis followed by detection with the aforementioned antibodies.31. Puromycin, is an aminonucleoside antibiotic which is an aminoacyl tRNAs structural analog that is incorporated into the nascent polypeptide chain and prevents its elongation. At low concentrations puromycin incorporation is directly proportionate to the rate of mRNA translation. Immunoblotting with the 12D10 monoclonal antibody to puromycin (Millipore) was used to detect puromycin incorporation. The translation inhibitor cycloheximide  used as a negative control. Cycloheximide was added to the cells 30 min prior to the puromycin addition. For puromycin labeling the cells were incubated at 37 °C with medium containing 10 µg/ml puromycin for 30 min. The cells were washed with PBS and incubated with fresh media for an additional hour followed by Western blot.Puromycin incorporation was used to monitor protein synthesisCells were cultured on coverslips, fixed for 30 min with 2% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and blocked with PBS + 1%BSA + 0.05% Triton for 1 h. Immunolabeling was performed overnight with the anti-viperin primary antibody (1:200). The secondary antibody was Alexa Fluor 555 goat anti-mouse (1:2000). 4′,6-diamidino-2 –phenylindole  was used for nuclei staining. Images were acquired using a Zeiss LSM800 confocal laser-scanning microscope  hooked to an inverted microscope.50. Briefly BSR-T7 cells grown on cover slips were left untreated or infected with VSV at the MOI of 3. For viperin experiments the cells were infected with a viperin and RFP expressing lentivirus and transfected with T7-GFP or vector control 24 h later. Following infection or transfection (24 h) the cells were depleted of uridine, by a 1 h incubation in low glucose DMEM (Invitrogen) supplemented with 20 mM D-glucosamine (Sigma). The cells were then transfected with 0.5 mM BrUTP (Sigma) incubated at 37 °C for 1 hour to allow the BrUTP incorporation. After the incubation, the cells were washed with PBS, fixed with 2% PFA, followed by 3 min ice-cold methanol, permeabilized and stained as previously described. RNA was detected using an antibody against bromodeoxyuridine . To inhibit cellular transcription cells were treated with 10 µg/ml actinomycin D  30 min before to BrUTP labeling. The quantitative analysis was performed using the ImageJ. Any cell displaying triple staining of red (viperin or luciferase control), GFP and blue (brU) was analyzed. The fluorescence intensity (FI) was calculated by multiplying the area of region of interest  by the average fluorescence intensity of this region. The cytosolic fluorescence intensity was calculated by subtraction of nuclear FI from total cell FI.5-Bromouridine experiments were performed as describedNo datasets were generated or analyzed during the current study.Supplementary Information


Breast fibromatosis is a rare disease characterized by monoclonal fibroblast proliferation. It has no ability to metastasize but has a high local recurrence rate and often infiltrates surrounding tissues. Surgical treatment is the reference, but recently, new targeted therapies have emerged. We report an original case of a patient with breast fibromatosis who received exclusive medical treatment. Our aim was to analyze these treatments based on the clinical and radiological outcome, iatrogenic effects, and pharmacological action.3). Initial surgery was not possible because of potential esthetic and functional prejudice. Thus, she had an exclusive medical treatment including several lines: NSAIDs with tamoxifen and triptorelin, followed by sorafenib, then interferon α2b, and finally sunitinib. With tyrosine-kinase inhibitors (TKIs) (sunitinib), a significant partial response was observed . For each treatment, we provided the clinical and radiological outcome in association with known pharmacological action.We report the case of a 19-year-old woman who developed a desmoid-type fibromatosis of the lower inner quadrant of the right breast, measuring 50 × 25 mm (i.e., a volume of 27.4 cmTKI had been an interesting alternative option to initial surgery, providing at least a partial response and potentially allowing less mutilating surgery. However, no pharmacological mechanism can unequivocally explain TKI efficacy. In general, breast fibromatosis should be treated along with oncologist and interventional radiologists in a trans-disciplinary modality, thus offering an adapted treatment for this particular desmoid-type fibromatosis localization. Fibromatoses (formerly desmoid tumor) are clonal fibroblast proliferations that develop in the deep soft tissue. One of their characteristics is their tendency to local recurrence, without the ability to metastasize. These lesions are usually poorly confined and infiltrate the surrounding tissues. Fibromatoses are classified into three groups according to the WHO: fibromatosis of the abdominal wall (AF), extra-abdominal (EAF), and intra-abdominal (IAF) .IAF is linked to familial adenomatous polyposis while both AF and EAF often occur sporadically. Etiology of these lesions remains uncertain: genetic mutations, trauma, hormonal factors, etc., have been mentioned. The incidence of sporadic fibromatosis (AF and EAF) ranges from two to four cases per million people –4. EAF aClinically, breast fibromatosis presents as a palpable, firm mass that may adhere to the chest wall, sometimes associated with skin retraction. According to the French National College of Gynecologists and Obstetricians (CNGOF), there is neither sufficient data to recommend surgery over conservative treatment nor optimal follow-up modalities and timing . The oveBefore 2000, most breast fibromatoses were surgically removed. Better understanding of the biology of these tumors and the introduction of new drugs (sunitinib (Sutent®), sorafenib (Nexavar®)) have enabled the development of medical protocols using targeted therapies. Few clinical studies evaluated targeted therapies efficacy in EAF; consequently nowadays, no guidelines are available.We report an original case of a patient with breast fibromatosis who received exclusive medical treatment. Our aim was to analyze these treatments based on the clinical and radiological outcome, iatrogenic effects, and pharmacological action, as an alternative to initial surgery.In October 2012 at the age of 19, Ms. L.E., nulliparous, with no previous medical history, was examined for breast pain and lump in the lower inner quadrant of the right breast. She reported that the mass appeared in 2009 and has slowly grown in size. Clinical examination confirmed the presence of a hard, ill-defined mass involving the pectoral muscle, associated with skin retraction. There was no suspicious axillary node.3 in volume , protein S100, CD34, calretinin, CD117, and p53. Some cells were expressing smooth muscle α-actin, and nuclear staining with anti β-catenin antibody was noted. Ki67 was estimated at 5%. These results confirmed the diagnosis of breast fibromatosis. After multidisciplinary discussion, we opted for an initial medical treatment, because the depth of muscular involvement increased esthetic and functional risks of surgery.3, Fig. 3, Fig. 3, Fig. 3, Fig. 3, Fig. 3, Fig. 3  plus tamoxifen , under cover of a GnRH agonist (long-acting triptorelin 3.75-mg intramuscular injection every 28 days), to prevent the risk of ovarian cysts linked to tamoxifen. In the months following the initiation of treatment, stabilization of tumoral volume and decrease in skin retraction were observed. Breast MRI at 5 months showed stable tumoral size . In our case, immunohistology study showed intranuclear accumulation of β-catenin, which may be present in up to 82% of breast fibromatosis . The WntInterferons (IFNs) are cytokines secreted by leukocytes (IFN-α) and fibroblasts (IFN-β). Once bound to their receptor (IFNAR-1), they activate in particular the JAK/STAT pathway that regulates expression of response genes having antiproliferative functions. Several cases of complete remission upon treatment with IFN-α have been reported in patients affected by limbs , 32 and Tyrosine-kinase inhibitors (TKIs) interact competitively with adenosine triphosphate to block phosphorylation of the intracellular tyrosine-kinase sites. The use of TKI is based on the overexpression of target proteins in tumors and their stroma. PDGFR is a receptor of the tyrosine-kinase family. After activation by its ligand (PDGF), it dimerizes and initiates a signaling cascade involving in particular the PI3K pathway, resulting in proliferation and cell differentiation. Vascular endothelial growth factor receptor (VEGFR), another tyrosine-kinase receptor, is a key pro-angiogenic factor. Type 1, 2, and 3 VEGFR are located on endothelial cells, and when activated, they cause the migration and proliferation of these cells. Proto-oncogene C-Kit (CD117), which belongs to the same family, links the stem cell growth factor and is a therapeutic target as well.Several TKIs are available, and imatinib (Gleevec®) is the most commonly used in fibromatosis. It targets C-Kit, PDGF, and Bcr-Abl. Imatinib was not used in our case because it was reported as resistance phenomena . SorafenOur patient is now asking about a potential pregnancy. Breast fibromatosis affect young women, and pregnancy is a particularly problematic issue. A study of Fiore et al., including 92 pregnant patients suffering from fibromatosis, showed that in 48% of cases, the onset of fibromatosis was related to pregnancy: the diagnosis was made either during pregnancy or 6 months after childbirth. Otherwise, 52% of the patients already had a history of fibromatosis, which was either clinically evident during pregnancy or had appeared before but already treated. The risk of tumor progression is high during or after pregnancy, even for patients already treated . Our patAs shown in our patient, medical treatments have heterogeneous efficacy. Targeted therapies may be a serious option to consider, especially when surgery is considered as high risk, thus leading to a less extensive surgery and a better functional and esthetic result. However, no pharmacological mechanism can unequivocally explain TKI’s efficacy. Besides medical treatments, other therapies are in development, such as cryotherapy. A trans-disciplinary approach is essential when dealing with desmoid-type mammary fibromatosis, joining targeted therapies to surgery/cryotherapy.


Stimulant medication increases learning rates and enhances performance in ADHD participants compared to effects in controls.Novelty seeking is characteristic of ADHD, but how this influences reward-related decision-making is unknown. Sethi  P = 0.027), and greater selection of novel options (P = 0.004). Moreover, persistence in selecting novel options predicted impaired task performance (P = 0.025). These behavioural deficits were accompanied by a significantly lower learning rate (P = 0.011) and heightened novelty signalling within the substantia nigra/ventral tegmental area (family-wise error corrected P < 0.05). Compared to effects in controls, stimulant medication improved attention deficit hyperactivity disorder participants’ overall task performance (P = 0.011), increased reward-learning rates (P = 0.046) and enhanced their ability to differentiate optimal from non-optimal novel choices (P = 0.032). It also reduced substantia nigra/ventral tegmental area responses to novelty. Preliminary cross-sectional evidence additionally suggested an association between long-term stimulant treatment and a reduction in the rewarding value of novelty. These data suggest that aberrant substantia nigra/ventral tegmental area novelty processing plays an important role in the suboptimal reward-related decision-making characteristic of attention deficit hyperactivity disorder. Compared to effects in controls, abnormalities in novelty processing and reward-related learning were improved by stimulant medication, suggesting that they may be disorder-specific targets for the pharmacological management of attention deficit hyperactivity disorder symptoms.Computational models of reinforcement learning have helped dissect discrete components of reward-related function and characterize neurocognitive deficits in psychiatric illnesses. Stimulus novelty biases decision-making, even when unrelated to choice outcome, acting as if possessing intrinsic reward value to guide decisions toward uncertain options. Heightened novelty seeking is characteristic of attention deficit hyperactivity disorder, yet how this influences reward-related decision-making is computationally encoded, or is altered by stimulant medication, is currently uncertain. Here we used an established reinforcement-learning task to model effects of novelty on reward-related behaviour during functional MRI in 30 adults with attention deficit hyperactivity disorder and 30 age-, sex- and IQ-matched control subjects. Each participant was tested on two separate occasions, once ON and once OFF stimulant medication. OFF medication, patients with attention deficit hyperactivity disorder showed significantly impaired task performance ( Attention deficit hyperactivity disorder (ADHD) is an early-onset neurodevelopmental disorder characterized by symptoms of inattention, impulsivity and/or hyperactivity with symptoms persisting into adulthood in up to 50% of patients , completion of the Conners’ ADHD self-report long version and Wender Utah questionnaires, informant history and wherever possible review of school reports. All had DSM-IV confirmed diagnoses of ADHD. Control participants were recruited through classified advertisements and university mailing lists. Participants gave written informed consent following full explanation of the experimental procedures. Local and national ethical approvals were obtained from Brighton and Sussex Medical School  and the East of England (Hertfordshire) National Research Ethics Committee (reference: 12/EE/0256).Exclusion criteria included past or current history of any neurological or psychiatric history, other than anxiety and/or unipolar depressive disorder currently in remission, past history of significant head injury, and current drug or alcohol abuse. Controls were additionally excluded if they had a history of serious cardiovascular conditions including cardiomyopathy, coronary artery disease, heart failure, ventricular arrhythmia or hypertension, current or recent use of monoamine oxidase inhibitors, coumarin anticoagulants, anticonvulsants or antipsychotics or a diagnosis of glaucoma. Of note, ADHD participants were routinely screened for these potential contra-indications to stimulant medication at clinical assessment.F = 0.20, P = 0.66], IQ , gender and handedness (ADHD and control participant were matched on age .To enable inference at the group level, the coefficient estimates for the model-based regressor  (3). Results are reported for clusters surviving a cluster forming threshold of P < 0.001 and a stringent family-wise error (FWE) extent threshold of P < 0.05 for the whole brain or appropriate region of interest.Bilateral ventral striatum and substantia nigra/ventral tegmental area were defined as post hoc t-tests. Non-parametric Spearman’s rho was used to assess relationships between behavioural measures to account for non-normal distributions within the data.The Conners self-report Adult ADHD Rating Scale CAARS;  were useα, β and total amount won on the task were affected by group or medication status using separate mixed-measures ANOVAs with group (between-subject) and medication (within-subject) factors. Planned analyses of effects of group and medication on novelty were assessed through inclusion of an additional within-subject novelty factor . To assess for long-term effects of medication on the behavioural novelty bonus, we also performed planned Spearman’s rho correlations between the novelty bonus and length of time on medication in months. Results of the above analyses were then further investigated using several post hoc behavioural tests as detailed in the relevant results sections.Planned analyses assessed whether the model parameters F = 17.01, P < 0.001. STAI trait: ADHD = 53.5 ± 11.0, control s = 36.5 ± 10.7, F = 36.51, P < 0.001], though group differences in antidepressant use were not significant . BDI and STAI scores did not significantly correlate with variables of interest .Mean (±SD) equivalent daily dose of methylphenidate was 50.0 ± 21.0 mg and mean psycho-stimulant treatment time 32.7 (±39.6) months for the ADHD group see . ADHD paF = 15.29, P < 0.001; harm-avoidance: ADHD = 17.1 ± 7.5, controls = 11.9 ± 7.5, F = 15.29, P = 0.01], but not reward-dependence  or persistence .Consistent with larger population studies  = 5.17, P = 0.027}. Moreover, stimulant medication had significantly different effects across the two groups (Group × Drug interaction), significantly enhancing ADHD participants’ performance compared to effects on controls who showed an inverse pattern of effects . Post hoc t-test confirmed a trend towards increased performance following stimulant medication in the ADHD participants  and a converse trend towards decreased performance in controls .OFF medication, ADHD patients showed impaired performance on the reinforcement-learning task (task performance) compared to controls {amount won [mean ± standard error (SE)]: ADHD: £86.3 ± 1.76, controls: £91.8 ± 1.80, nQ = fQ), and found that it did . Consistent with what we observed for task performance, unmedicated patients with ADHD showed a significantly lower learning rate than unmedicated controls . Stimulant medication also demonstrated dissociable effects on learning rates across groups  significantly increasing learning rates in ADHD  compared to effects on controls . However, though differences in effects of stimulant medication could be observed between groups, these did not survive within group comparisons (post hoc t-test P > 0.1). Choice-randomness (β) did not significantly differ between groups or across medication condition   .Table 2Qn > Qf; novelty bonus = £0.039 ± 0.01; F = 10.84, P < 0.005; see Qf and Qn for each condition and group separately]. Indeed, unmedicated ADHD participants expressed a novelty bonus more than double that observed in controls (£0.054 ± 0.018 versus £0.024 ± 0.015), though due to high interindividual variability this effect did not reach statistical significance .Across groups, unmedicated participants showed a strong preference for novel compared to familiar stimuli:  with post hoc analysis indicating a heightened salience of intrinsically ‘novel’ stimuli rather than an increased propensity to choose all newly introduced stimuli .To investigate whether group differences in novelty preference were expressed at the behavioural level, we then examined how frequently individuals selected novel compared to familiar stimuli on their first appearance. Unmedicated ADHD participants were significantly more likely than controls to choose novel compared to familiar options on their first presentation . As anticipated, better performance ON medication was associated with a lower (i.e. more accurate) initial valuation of both novel (Qn) and familiar (Qf) stimuli  or non-optimal .F = 10.04, P = 0.002]. Neither medication nor group status showed main effects on this. However, medication did differentially affect how long the two groups selected optimal versus non-optimal novel options   = 7.60, P = 0.010]. However, this pattern of effects was not observed in controls . Across groups, participants also showed the expected tendency to persist with optimal rather than non-optimal familiar options . However, medication did not differentially affect how long the two groups selected optimal versus non-optimal familiar options . This indicates that stimulant medication selectively enhanced ADHD participants’ accuracy in discriminating optimal from non-optimal novel but not familiar options. This may reflect a steeper decay in the additive value of novelty induced by stimulant medication in the context of ADHD, which served to optimize decisions directed toward non-familiar (i.e. novel) stimuli.Across groups, participants showed a greater tendency to persist with optimal rather than non-optimal novel options [= 0.032] . BreakinF = 1.59, P = 0.213]. Previous studies show long-term alterations in striatal dopamine availability following sustained methylphenidate use (P = 0.018), i.e. patients treated the longest showed the lowest novelty bonuses.Despite having a mean novelty bonus more than double that of controls, ADHD patients showed marked interindividual differences that overshadowed the statistical significance of group effects  with a significant first (P = 0.005) but not second (P = 0.126) pair of canonical variates. The first novelty/reward derived canonical variate explained 63% of variance in the ADHD clinical phenotype canonical variate and 41% of overall ADHD phenotype . The ADHD canonical variate was highly loaded by inattention scores (r = 0.99) and moderately loaded by hyperactivity/impulsivity scores (r = 0.54). The novelty/reward canonical variate was moderately loaded by OFF (r = −0.37) and ON medication learning rate (r = 0.36), ON medication amount won (£) (r = 0.53) and % novel items picked on first appearance (r = −0.65) and near-moderately loaded by OFF medication novelty bonus (r = 0.29) . Overall, these data provide evidence that novelty/reward-related behavioural features explain up to 41% of the variance in clinical ADHD phenotype.Canonical correlation analysis was used to examine how reward and novelty related behavioural features  (set 1) related to clinical phenotype (inattention and hyperactivity/impulsivity domains of ADHD) (set 2). Briefly, this analysis derived latent canonical variates from linear combinations of each of the two sets of variables, to maximize their covariance. Results demonstrated a significant model [Wilk’s Lambda test: base) showed a tight correlation (FWE: P < 0.05) with bilateral ventral striatum and orbitofrontal cortex activity  across groups ], where the ADHD group exhibited a significant reduction in neural signals encoding reward prediction error while on stimulant medication compared to placebo. The opposite pattern was observed in controls ] indicating a significant reduction of novelty-related signalling in ADHD participants ON stimulant medication compared to placebo, and a converse pattern observed in controls , but not ventral striatal reward prediction error signalling  were related to improved task performance.Finally, we sought to investigate whether drug-related reductions in baseline ventral striatal reward prediction error signalling or substantia nigra/ventral tegmental area novelty processing best explained the drug-induced enhancement of performance in ADHD. Consistant with our behavioural findings, drug-induced reductions in substantia nigra/ventral tegmental area novelty-bonus signalling  stimuli on their first presentation. This tendency was captured computationally as a higher  novelty bonus and a significantly lower rate of value updating in response to reward (lower learning rate). This heightened salience of novelty, coupled with a slower decay in its rewarding properties, served to bias ADHD patients to repeatedly select novel options even when they were non-optimal. Interestingly, stimulant medication selectively remediated many of these abnormalities in ADHD compared to effects in controls. For example, compared to effects in controls, stimulant medication improved ADHD participants’ overall task performance, reduced their reward-learning rates and enhanced their ability to differentiate optimal from non-optimal novel choices. Aberrant persistence with selecting novel stimuli in the drug-free state additionally predicted ADHD participants’ response to treatment. These findings were complimented by our brain imaging data that showed that stimulant medication resulted in a significant reduction in substantia nigra/ventral tegmental area novelty-bonus signalling in ADHD participants, which significantly correlated with improved task performance. Finally, preliminary cross-sectional evidence suggested an association between long-term stimulant treatment and a reduction in the rewarding value of novelty. Together, these results highlight a central role for aberrant novelty processing in reward-related decision-making abnormalities observed in ADHD. Moreover, reward and novelty abnormalities appear to play an important role in the clinical phenotype of ADHD, with our canonical correlation analysis revealing up to 41% of the variance in clinical features of ADHD being explained by differences in reward and novelty processing.Previous modelling with simulated data has predicted that hypo-dopaminergic abnormalities will reduce learning rates and in turn account for key components of impulsive reward dysfunction in ADHD . By showAt first glance, the increased novelty and reward prediction error signals we observed in ADHD may appear at odds with the hypo-dopaminergic profile believed to be central to this disorder. However, this divergence is predicted by a number of accounts of ADHD, which suggest that despite a reduction in tonic dopamine, phasic dopamine release is likely increased . One posIn contrast to the beneficial effects we observe in ADHD , methylphenidate did not significantly affect behavioural responses to novelty in controls. Interestingly, previous work has shown that in other, broader cognitive domains, methylphenidate has similar effects in both ADHD patients and controls ]. Though this indicates that our prediction error results are unlikely to be substantially contaminated by differences in value signals, it did mandate that like Wittmann, our primary outcome variable (novelty bonus) was a combination of Qadd and δadd. Finally, our data were acquired at a field strength of 1.5 T. Though signal-to-noise will have been enhanced by our use of a 32-channel head coil, a higher performing system may have been sensitive to smaller effects in regions we have not reported.This work must be interpreted in the light of several limitations. First, we report results from a sample with prior exposure to stimulant medication. Though this was done to ensure a sample in which these medications were clinically efficacious, differential effects of medication observed across groups may have been influenced by differences in prior exposure. Second, our task was designed to mirror that of Further work is required to consolidate the broader clinical implications of the aberrant responses to novelty observed here. For example, in addition to apparent roles in inattention and poor decision-making, heightened novelty valuation could well contribute to the high prevalence of substance use disorders observed in ADHD. Conversely, the apparent reduction in novelty valuation we observe with prolonged treatment could underlie the reported reduction in substance abuse risks associated with long-term medication use (Supplementary DataClick here for additional data file.


People living on the high plateaus of the world have long fascinated biological anthropologists and geneticists because they live in "thin air" and epitomize an extreme of human biological adaptation. Far from posing obstacles to human habitation, the hypoxic conditions of the Tibetan Plateau, the Andean highlands, and the Amhara Plateau of Ethiopia have served as the contexts for natural experiments in human adaptability . To the GCH1 (GTPcyclohydrolase Ⅰ) gene and the EP300 (histone acetyltransferase p300) gene, which are both involved in maintaining nitric oxide synthetase (NOS) function and normal blood pressure, harbor potentially adaptive variants in Tibetans. Both genes show high allelic divergence between Tibetan and lowland-dwelling Han Chinese, with signals of positive selection in the Tibetans.Among the most dramatic adaptations to hypobaric hypoxia seen in high-altitude-dwelling Tibetans are the higher levels of the vasodilator, nitric oxide, in their exhaled breath and blood. Regulation of the pulmonary vascular response to hypoxia has been recognized as a key feature of the high altitude adaptation of Tibetans, but until recently little was known of the genetic basis of this phenomenon. Two recent papers in this journal from the laboratory of Su Bing, by These discoveries add to the extensive catalogue of evidence indicating that the Tibetan genome has undergone natural selection at multiple loci concerned with distinct aspects of blood-related phenotypes, including regulation of hemoglobin phenotype  and fola