Background: Attention-deficit/hyperactivity disorder (ADHD) is a disabling disorder. High rates of ADHD have been consistently reported among prisoners. The main objectives were (1) to estimate the prevalence of ADHD symptoms in a sample of male inmates and (2) to investigate the relationship between ADHD symptoms and socio-demographic/clinical features. According to the high prevalence of childhood trauma among inmates, we assessed whether exposition to childhood trauma can be related to the presence of ADHD symptoms. Methods: A total of 159 male prisoners admitted to Monza prison between January 2020 and June 2021 were included. Both Wender Utah ADHD rating scale and adult ADHD self-report scale were administered to assess ADHD symptoms. Moreover, inmates completed the childhood trauma questionnaire. Results: Data were available for 108 inmates. Thirty-five prisoners (32.4%) were found on screening to meet the criteria for symptoms of ADHD. Cocaine use disorder, prescription of mood stabilizers and a history of emotional abuse significantly increased the likelihood of having clinically significant ADHD symptoms. Furthermore, patients who experienced physical neglect resulted in meeting the criteria for ADHD symptoms. Conclusions: ADHD symptoms are widespread among inmates and are associated with specific risk factors. Screening for ADHD should be done to provide appropriate intervention strategies. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition DSM-5), Attention-deficit/hyperactivity disorder (ADHD) is defined as a persistent pattern of inattention and/or hyperactivity/impulsivity that interferes with functioning or development. A level of impairment in a minimum of two areas of life needs to be manifest for at least six months to make diagnosis of ADHD . Moreove, AttentiEven though there was increasing sensitivity in the recognition and management of ADHD in the community, few studies focused on this topic with regard to subjects hosted in detention centers. Nevertheless, the relationship between ADHD and other psychiatric comorbidities, substance use disorders, and childhood trauma is still poorly studied in prison populations. With our study, we firstly aimed to investigate the prevalence of ADHD symptoms among a sample of adult male prisoners. Secondly, we aimed to find potential predictors of clinically significant ADHD symptoms for incarcerated male subjects with a specific focus on the different types of coexisting mental disorders (including substance use disorders) and childhood trauma. As discussed above, there is an urgent need to identify adult offenders with ADHD symptoms and to intervene proactively. Indeed, we hypothesized that ADHD is prevalent among prisoners and that subjects with ADHD symptoms have more comorbid mental disorders and substance misuse compared to the ones without ADHD symptoms. Moreover, we expected that one or more types of childhood trauma might better discriminate between the ADHD and non-ADHD groups, thus increasing knowledge regarding this condition. These aspects are of current interest because prisoners with ADHD symptoms are frequently undiagnosed, and they may be treated for a comorbid psychiatric condition instead of their underlying ADHD ,22,23.This is a cross-sectional study of consecutive newly arrived inmates admitted to Monza prison , between January 2020 and June 2021. At present, the prison houses only male offenders. Newly arrived prisoners eligible for this study were on remand or sentenced, admitted in the penitentiary for the first time or formerly incarcerated, aged between 18 and 65 years, and they expressed a willingness to participate in the study. We excluded prisoners unable to speak or read Italian or with too severe cognitive impairment, learning disabilities, and psychiatric symptoms to complete the study. Moreover, participants with any missing data were excluded. A specifically trained team of psychiatrists and psychologists conducted a clinical interview within the first 72 h after the incarceration. Indeed, in most adult prisons newly arrived offenders are subject to a reception screen upon admission aimed to review their overall mental health problems, including ADHD, as well as detecting the risk of self-injurious behaviours and suicidality .All data were anonymously collected and registered in an appropriate dataset, not allowing patients’ identification. Self-reported measures included age, nationality , partnership (single/divorced/widowed versus partner/married), education level (low-medium versus high), unemployment (yes versus no), and previous incarceration (yes versus no). Moreover, participants were asked about history of nonsuicidal self-injuries (NSSIs) and attempted suicide in and out of prison. NSSI is defined as the deliberate, direct, socially unacceptable destruction or alteration of body tissue . On the other hand, we considered as ‘suicide attempts’ only self-injuries of people willing to die . AlthougThe prescription of psychotropic medications was defined by the use of at least one of the following drugs: antipsychotics, mood stabilizers (lithium and anticonvulsant agents), and antidepressants, as well as anxiolytics or hypnotics. We included psychotropic medications prescribed both before imprisonment  and within the first 72 h after incarceration. In this regard, psychotropic medications could have been prescribed by either psychiatrists or other medical practitioners working in the prison. Information about comorbid medical conditions  was acquired according to medical records or patients’ self-report.ADHD symptoms were assessed by asking adults to complete both the Wender Utah ADHD rating scale (WURS) and adult ADHD self-report scale (ASRS). The 25-item version of the WURS was used to retrospectively evaluate ADHD-related symptoms in childhood . The aduSubstance use and other mental disorders were assessed according to the diagnostic criteria of the DSM-5. Drug-specific diagnoses for the following substances were included: alcohol, cannabis, cocaine, heroin, hallucinogens, ecstasy, ketamine, inhalants, prescription opioids, sedatives or tranquilizers, and stimulants. Each DSM-5 substance use disorder diagnosis required fulfilling two or more of the 11 criteria in the 12 months preceding the interview .History of childhood trauma was evaluated by the 34-item CTQ . The CTQp < 0.05, together with age and previous incarceration, were entered as independent variables into a logistic regression model with ADHD-P as the dependent variable. Adjusted odds ratios (aOR), together with their 95% confidence intervals (CI), were reported.Descriptive analyses of included variables were performed for the total sample: mean and standard deviation (SD) for quantitative variables and frequency and percentage for qualitative ones. The total sample was divided into ADHD-screened positive (ADHD-P) and ADHD-screened negative (ADHD-N) groups according to the cut-off levels for WURS-25 and ASRS-V 1.1, respectively. First, we carried out univariate analysis in order to detect statistically significant differences in these two groups. The normal distribution of quantitative variables was verified by using Shapiro–Wilk’s test. Age was compared between groups by independent sample T test. The groups were compared for qualitative variables by chi-square tests. Finally, all variables from the univariate analysis with Analysis was conducted using Stata Version 13.1 SE .Our original sample consisted of 159 inmates. Among these, 38 were excluded because they did not meet inclusion criteria: 16 did not speak or read Italian; 5 suffered from learning disabilities; 17 individuals had severe psychiatric symptoms not allowing them to take part to clinical interview. Moreover, 13 were excluded in view of active refusal or incomplete data. Therefore, data were available for 108 male inmates. A flow chart of selected subjects is reported in According to the cut-off levels for both WURS-25 and ASRS-V 1.1, thirty-five prisoners (32.4%) were considered ADHD-P. The majority of our sample (57.0%) was previously incarcerated. Personality disorders (45.5%) were the most common diagnosis, followed by psychotic disorders (11.2%) and depressive and adjustment disorders (11.1%). The highest 12-month prevalence rates of substance use disorders were for cocaine (73.1%), cannabis (60.2%), and alcohol (33.3%). Unlike NSSI (25.0%), most individuals reported to have attempted suicide out of prison (9.3%). Anxiolytics or hypnotics (67.6%) and antipsychotics (35.2%) were the most commonly prescribed psychotropic medications in the whole sample. The most-experienced childhood trauma was emotional (20.4%) and physical (19.4%) abuse .Age was statistically lower in the ADHD-P group. Prisoners with a diagnosis of personality disorder, as well as of cocaine use disorder, were more likely to be ADHD-P. ADHD-P subjects had higher prevalence of NSSIs in prison. The ADHD-P group reported a higher rate of referral to outpatient child and adolescent psychiatry services as compared with the ADHD-N group. Moreover, a higher rate of the prescription of mood stabilisers and anxiolytics or hypnotics was found among ADHD-P prisoners. With regards to childhood trauma, a history of emotional abuse and physical neglect was associated with the presence of ADHD-P.p = 0.020) and prescription of mood stabilisers , as well as a history of emotional abuse   .n = 90) who were assessed within 10 days of admission to the detention centre met the criteria for ADHD in childhood, and 60% of those were either fully symptomatic or in partial remission of their symptoms. On the other hand, a study conducted on a sample of 194 adult male longer-term prison inmates by Ginsberg et al. [Apart from a pilot study conducted on a sample of 59 Italian inmates , to the g et al.  reportedg et al. . Indeed,g et al. .Beyond the high prevalence of ADHD-P within our sample of male prisoners, we found three main results. First of all, we observed that ADHD-P group was more likely to have cocaine use disorder than ADHD-N one. Different hypotheses have been proposed to clarify the strong relationship between substance use disorder and ADHD. Familial studies confirmed a shared genetic substrate between ADHD and substance use disorder . MoreoveSecond, we found that ADHD-P prisoners were more likely to receive the prescription of a mood stabiliser than ADHD-N prisoners. ADHD can present with irritability, emotional dysregulation, mood lability, low self-esteem, low frustration-tolerance, and sleep problems, favouring misdiagnosis with other mental conditions such as personality disorders and mood disorders . NeverthThird, childhood trauma was significantly related to the presence of ADHD-P. To the best of our knowledge, this is the first study exploring the possible connection between childhood trauma subtypes and ADHD among prison inmates. Even though multiple factors contribute to the etiology of ADHD, a growing body of research has hypothesized a relationship between ADHD onset and early stressful life events, more specifically childhood trauma, in both children and adult samples. Indeed, different studies indicated that childhood trauma occurring prior to young adulthood may be more common among individuals with ADHD in comparison to non-ADHD groups, and greater levels of ADHD symptoms may be found among people who were exposed to childhood trauma compared to non-exposed subjects . HoweverFinally, despite less robust association than results statistically significant in the final logistic regression model, patients diagnosed with personality disorder and history of NSSIs are more frequently ADHD-P. To date, it is widely recognized that prisoners with ADHD symptoms have an increased risk of comorbid disorders. According to a meta-analysis of 18 studies including 1615 individuals with ADHD and 3128 without ADHD, adult prisoners with ADHD had a 3-time higher risk of personality disorders compared to the counterpart . MoreoveIn any case, according to best practices, all new prisoners should receive a first reception screen for ADHD. In particular it is important to review the medical history and evaluate the presence of other common mental or neurodevelopmental disorders that may overlap with ADHD—emphasising the need for a careful diagnostic assessment . As a reThere are some important methodological limitations related to the present study. First, as this study was a cross-sectional one, we cannot definitively define whether the independent variables preceded or followed the ADHD symptoms. Secondly, the self-report scales, despite appropriate for detecting ADHD symptoms, are less reliable to make a diagnosis of the disorder compared to semi-structured interviews. Indeed, the requirement to be positive for ADHD in two different rating scales increases the reliability, but it still does not totally remove potential recall bias . TherefoDespite some limitations, the current study shows that ADHD symptoms are common among newly arrived male prisoners. In particular, cocaine may be related to a higher risk of ADHD symptoms than other substances. Moreover, ADHD-P subjects are more likely than the counterpart to have been prescribed mood stabilisers. Therefore, it is likely that ADHD may be overlooked and is often seen as secondary to different apparently more important conditions, such as mood disorders or substance misuse . Of note


The number of individuals who are diagnosed with attention‐deficit hyperactivity disorder (ADHD) during adulthood has increased in recent years. However, there is still no decision aid (DA) to help adults newly diagnosed with ADHD make decisions regarding further treatments.This study aimed to describe the development process of a DA for adults newly diagnosed with ADHD and its field testing during the shared decision‐making (SDM) process in a clinical setting.The development process involved the creation of a DA prototype using the International Patient Decision Aid Standards criteria and revision of the prototype through the stakeholders' reviews. The field testing of the DA compared scores before and after the SDM process on the service users' knowledge scale, decisional conflict scale and the Conners Adult ADHD Rating Scales.The developed DA contained options of watchful waiting with own coping skills and pharmacological treatment, which consisted of several kinds of drug options. Fifteen adults newly diagnosed with ADHD participated in the field testing. The participant decision‐making quality outcomes such as their knowledge and decisional conflict improved after the SDM process. ADHD severity did not change.A DA for adults with ADHD was systematically developed following the international criteria. Field testing indicated that the DA could serve as a tool to facilitate the SDM process. Further research on this DA is necessary before its routine implementation.During the development process of the DA, the service users who had already been diagnosed with ADHD reviewed the DA prototype and provided feedback, which improved the final version of the DA. We also assessed if the DA, which included examples of behavioural changes to cope with ADHD symptoms, would reduce ADHD severity.The DA developed in this study is anticipated to improve the quality of initial care for adults newly diagnosed with ADHD. This service user involvement approach might have a potential positive impact on the abilities of these adults to cope with ADHD symptoms.22.1The content of a DA prototype was incorporated by findings from the qualitative need's assessment interviews, which were already reported elsewhere,2.2The developed DA was then field‐tested during the SDM process in a clinical setting described below.2.2.1We conducted field testing of the DA using a clinical SDM process that we developed and reported previously.Initial consultation: Option presentation consultation: After the psychiatrist informs the adult of the diagnosis as ADHD, further treatment options such as watchful waiting with coping skills or additional medication with coping skills are shared between the adult and the psychiatrist using the DA booklet.Step 1. Deliberation outside the service: The participant goes home with the DA, and freely reviews and considers it.Step 2. Decision‐making consultation: At the next appointment, the participant sees the psychiatrist. Both parties discuss the treatment options using the DA and finally decide which treatment can be further taken by the participant.Step 3. 2.2.2Participants were recruited from three outpatient psychiatric services in Tokyo from March 2019 to February 2020. For the inclusion criteria, we included those who visited the psychiatric services and met the following criteria: (i) outpatients aged 20 years or older; (ii) received a first‐time ADHD diagnosis followed by Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (DSM‐5).2.2.3Baseline—filled the questionnaires immediately after the initial consultation(a) Preferences for roles in decision making: We used the Control Preference Scale (CPS)κ coefficients = 0.61).Decisional conflict: We assessed decisional conflict, defined as uncertainty about the course of action to take when choosing among several medical procedures,α = .84–.96).(b) Knowledge: Participants' knowledge regarding ADHD and its treatment was measured using a questionnaire containing 18 items with the response options ‘true’, ‘false’, and ‘don't know’. Of the 18 items, 13 items were presented in the DA and 5 items were not. The number of items, which were answered correctly within each subgroup, that is, the items presented in the DA and those not presented in the DA, was calculated. We developed this questionnaire for this study based on knowledge measures used in the previous ADHD study(c) ADHD severity: We assessed ADHD severity using Conners' Adult ADHD Rating Scales‐Self‐Report: Long version (CAARS‐S: L).α = .73–.88).(d) Post—filled the questionnaires immediately after decision‐making consultationHow decisions were made: We used the scales developed by Strull et al.(a) Decisional conflict: We assessed decisional conflict using the Japanese version of the DCS.(b) Knowledge: Participants' knowledge regarding ADHD and its treatment was measured using exactly the same questionnaire that was administered after the initial consultation.(c) One‐ to 2‐month follow‐up2.2.4ADHD severity: Within the 1–2‐month follow‐up after the decision‐making consultation, we assessed ADHD severity using the Japanese version of CAARS‐S: L2.2.51) was used to determine the concordance.We assessed the concordance between preferences for a role in decision‐making and how decisions were made. We combined the three categories reflecting participant involvement into one category and contrasted it with the categories of the clinician‐alone and patient‐alone decision‐making. Gwet's Agreement Coefficient  were also calculated.For the knowledge, decisional conflict and ADHD severity, we used a paired This study protocol was approved by the Ethics Board at St. Luke's International University (18‐A055).33.1The developed DA prototype consisted of a 20‐page A5 paper prepared as a booklet. The needs assessment interviews suggested that individuals diagnosed with ADHD during adulthood experienced difficulties in accepting their diagnosis and had identity concerns just after the diagnosis.Five service users then reviewed the DA prototype (Supporting Information Appendix 3.23.2.1Participants were 15 adults newly diagnosed with ADHD. The mean age was 42.4 years. Table 3.2.21 = 0.88 (0.60–1.09). This indicated that it was more than a coincidence.Regarding preferences, 14 (93%) of participants preferred patient involvement in decision making, while only 1 (7%) preferred clinician‐alone decision‐making and none preferred patient‐alone decision‐making. Regarding practical experiences, 14 (93%) reported that they were involved in the decision‐making, while only 1 (7%) answered that the clinician made the decision, and none chose patient‐alone decision‐making. The concordance between role preferences and experienced decisions (15 pairs) was AC3.2.3Decisional conflict: The participants' mean total decisional conflict score at baseline was 56.4 (±20.0). After the SDM process, the mean score was 32.8 (±14.4) (p = 0.001), which indicates that their conflicts significantly improved, and they were feeling more confident about their decisions. All subscales were >37.5 at baseline, indicating that the participants were feeling unsure about the implementation of each category ; however, they all significantly improved after the SDM process. The mean subscales, other than ‘uncertainty’, after the SDM process were <37.5 (Table (a) Knowledge: The participants' mean knowledge score of the specific items mentioned in the DA improved from 5.8 (±3.3) at baseline to 9.7 (±2.2) after the SDM process (p = 0.001). The participants’ mean knowledge score of the items not mentioned in the DA also improved after the SDM process (p = 0.003) (Table (b) rom 5.8 ±.3 at bas3.2.4There was no significant change between the baseline and the 1–2‐month follow‐up regarding any subscale Table .4To our knowledge, this is the first study to develop a DA for adults newly diagnosed with ADHD and to conduct field testing of the DA during the SDM process in a clinical setting.The findings indicate that adults with ADHD are able to participate in the SDM process while acquiring the relevant knowledge and assessing their preferences. The concordance between their role preferences and experienced decisions also indicated that adults with ADHD were successfully involved. Moreover, the significant decrease of decisional conflict, after the SDM process, suggested that the participants made effective decisions regarding further treatments. Ramon et al.,However, this study found no change in the ADHD severity in the 1–2‐month follow‐up. Several reasons may underlie this absence of change. The sample size of the study was small. Also, the 1–2‐month follow‐up might not be appropriate to adequately assess the ADHD severity because the participants were still struggling to acquire new strategies during that period. It takes a little longer for their coping skills to adapt to their environments. Brinkman et al., who conducted an SDM intervention for the parents of children with ADHD, also reported no significant difference in the behaviour rating scale for assessing treatment response.There were some limitations in this study. First, the sample size was relatively small because we recruited the participants in the services where the SDM programme had been introduced.Second, because of the exploratory nature of this study as a field testing, the evaluations were not compared with adults newly diagnosed with ADHD who had decided on further treatment without the DA, and who were not involved in treatment decision‐making. As the international criteria5We systematically developed a DA for adults newly diagnosed with ADHD following the international criteria. The field testing suggests that the successfully developed DA could serve as a tool to facilitate SDM in clinical settings. Further research on this DA is necessary before its routine implementation.The authors declare that there are no conflict of interests.All authors conceptualized and designed the study. Yumi Aoki secured the grant to conduct the study. Takashi Tsuboi, Yoshikazu Takaesu and Koichiro Watanabe organized data collection. Yumi Aoki carried out the questionnaire surveys. Yumi Aoki, Takashi Tsuboi, Kazuhiro Nakayama, Yasuhito Kinoshita and Mami Kayumi contributed to the analysis and writing of the manuscript. All authors read and approved the final draft.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


Objective: Attention deficit hyperactivity disorder (ADHD) is a commonly diagnosed neuropsychiatric disorder in children, which is characterized by inattention, hyperactivity and impulsivity. Using resting-state functional magnetic resonance imaging (R-fMRI), the alterations of static and dynamic characteristics of intrinsic brain activity have been identified in patients with ADHD. Yet, it remains unclear whether the concordance among indices of dynamic R-fMRI is altered in ADHD.Methods: R-fMRI scans obtained from 50 patients with ADHD and 28 healthy controls (HC) were used for the current study. We calculated the regional dynamic changes in brain activity indices using the sliding-window method and compared the differences in variability of these indices between ADHD patients and HCs. Further, the concordance among these dynamic indices was calculated and compared. Finally, the relationship between variability/concordance of these indices and ADHD-relevant clinical test scores was investigated.Results: Patients with ADHD showed decreased variability of dynamic amplitude of low-frequency fluctuation (dALFF) in the left middle frontal gyrus and increased one in right middle occipital gyrus, as compared with the HCs. Besides, ADHD patients showed decreased voxel-wise concordance in the left middle frontal gyrus. Further, lower voxel-wise concordance in ADHD’s left middle frontal gyrus was associated with more non-perseverative errors in Wisconsin Card Sorting Test, which reflects worse cognitive control.Conclusion: Our findings suggest that variability and concordance in dynamic brain activity may serve as biomarkers for the diagnosis of ADHD. Further, the decreased voxel-wise concordance is associated with deficit in cognitive control in ADHD patients. Attention deficit hyperactivity disorder (ADHD) is characterized by inattention, hyperactivity and impulsivity. It is one of the most commonly diagnosed childhood neurodevelopmental disorder, whose prevalence in school-age children and adolescents is estimated at 7.2% . This diMaladaptive in the intrinsic brain activity of ADHD was frequently reported in previous studies. Many of these findings were derived from resting-state functional magnetic resonance imaging (R-fMRI), which measures the blood oxygen level dependent (BOLD) signal without task or stimulation and further characterizes the spatiotemporal organization of brain activities . In specThese R-fMRI indices have been used to characterize the maladaptive brain functions in patients with ADHD. For example, children with ADHD exhibited increased ALFF in the occipital cortex and globus pallidus , and decAlthough these studies provided insights into the neural basis of ADHD, they failed to capture the dynamic changes in brain activities. Recent evidence demonstrated the temporal dynamic in brain activity patterns  and founIn addition, the concordance, i.e., temporal and spatial coupling, among multiple dynamic brain activity indices shows stable individual difference . StudiesThe current study aimed to bridge this gap. In specific, we compared the variability of dynamic R-fMRI indices and concordance among them between patients with ADHD and healthy controls, and further correlated the variability/concordance with the clinical test scores. Based on the results of previous studies, we hypothesized that ADHD patients would exhibit abnormalities in dynamic brain activity and its concordance, and this concordance would be related to its clinical manifestations. Our study may enhance the understanding of the neural mechanism underlying ADHD.Fifty-eight drug-naïve ADHD patients were recruited from the Out-patient Clinic of the First Affiliated Hospital of Wenzhou Medical University. Meanwhile, 31 healthy controls matched in gender, age and IQ were recruited from local schools. The diagnosis of ADHD was performed by three experienced psychiatrists according to the Fifth Edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). For all the participants, the exclusion criteria were: (1) conduct disorder, oppositional defiant disorder, or any other psychotropic disorder besides ADHD; (2) history of major neurological or physical illness; (3) current or history use of psychotropic drugs; (4) intelligence quotient (IQ) lower than 80; (5) MR scanning contraindications. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. All participants were well informed and written informed consent was obtained before the test.The revised version of Conners’ Parent Rating Scale (CPRS)  was usedThe computerized Wisconsin Card Sorting Test (WCST)  was usedImages were acquired in a 3T GE Sigma HDX 3.0 scanner  with the following parameters: repetition time (TR) = 2,000 ms, echo time (TE) = 30 ms, 31 axial slices, slice thickness = 4 mm, slice gap = 0.2 mm, flip angle = 90°, matrix size = 64 × 64, field of view (FOV) = 192 × 192 mm, and 240 volumes. High-spatial-resolution T1-weighted 3D images were obtained axially using the following parameters:162 axial slices, repetition time (TR) = 7,200 ms, echo time (TE) = 2.2 ms, flip angle = 7°, section thickness = 1 mm, gap = 0, field of view (FOV) = 256 × 256 mm, data matrix = 256 × 256. During the scanning, sponge earplugs were used to reduce scanner noise, and foam cushions were used to minimize head movement. The subjects were required to be relax, stay still, close their eyes, and stay awake.Data Processing and Analysis for Brain Imaging (DPABI)  was usedR-fMRI indices were calculated with following procedures:ALFF  of each index across the time series from each voxel was calculated and normalized across the voxels to measure the variability in these dynamic R-fMRI indices. Finally, the normalized SD maps of each dynamic R-fMRI index were smoothed (FWHM = 4 mm) to improve the signal-to-noise ratio.In particular, dynamics of these R-fMRI indices were analyzed using Temporal Dynamic Analysis (TDA) toolkits based on DPABI . SpecifiN = number of voxels) voxel-wise concordance values.The concordance of five dynamic R-fMRI indices  were calculated. In specific, volume-wise and voxel-wise concordance was measured by Kendall’s W among these indices . The volImportantly, we validated the results with the window length of 48 and 64 TR.t-tests. Meanwhile, gender distributions were compared using chi-square test. These statistical analyses were performed with SPSS 23.0.We compared age, IQ, ADHD-relevant clinical variables between ADHD patients and healthy controls using two-sample t-test, and the whole-brain gray mask was applied, and the results were corrected by multiple comparisons using Gaussian random field theory . Meanwhile, the volume-wise concordance was compared using analysis of covariance (ANCOVA). Further, Pearson or Spearman partial correlation was used to quantify the associations between dynamic indices and ADHD-relevant clinical test scores. Note that the age, gender, IQ, and head motions are controlled as covariates.The dynamic R-fMRI indices and voxel-wise concordance were compared using two-sample P = 0.014).The characteristics of samples were summarized in p < 0.001, cluster-wise p < 0.05, two-tailed; see Compared with HCs, the ADHD group exhibited increased dALFF variability in right middle occipital gyrus and decreased dALFF variability in left middle frontal gyrus (GRF, voxel-wise p < 0.001, cluster-wise p < 0.05, two-tailed; see The ANCOVA results indicated no significant differences in volume-wise concordance between the ADHD group and HC group. However, ADHD patients showed decreased voxel-wise concordance in left middle frontal gyrus (GRF, voxel-wise r = −0.367, p = 0.012; see r = 0.099, p = 0.646; see No significant correlations were found between the CPRS scores and altered dALFF variability for the ADHD group. However, the abnormal voxel-wise concordance of the left middle frontal was negatively correlated with the non-perseverative errors in WCST of patients with ADHD (Pearson partial correlation, The validation results with the window length of 48 and 64 TR showed good agreement with the results mentioned above .Abnormality in intrinsic brain activities is tightly associated with ADHD. This study systematically compared the dynamic changes in resting-state brain activities of patients with ADHD with HCs. Specifically, we calculated the temporal variability, spatial and temporal concordance of dynamic R-fMRI indices including ALFF, ReHo, DC, VMHC, and GSC in both groups. These properties were then compared between groups and correlated with the clinical variables, in which both the alterations and correlations were identified.Specifically, for the variability, ADHD patients exhibited increased dALFF variability in right middle occipital gyrus and decreased one in left middle frontal gyrus, which may reflect the hyper-activation in visual cortex and hypo-activation in frontal cortex. This is consistent with a previous study in which decreased ReHo was found in frontal and increased ReHo and ALFF was found in visual cortex .The development of the frontal lobe is essential for the acquisition, execution and control of a wide range of functions . And froStructural and functional alterations in occipital were also reported in previous studies. For example, reduced gray matter volume in the right middle occipital gyrus  and hypeAlterations in concordance among the dynamic R-fMRI indices were also identified in patients with ADHD. A recent study found that R-fMRI regional indices was high concordant in brain cortical and subcortical areas across the time window , and theIn addition, we found that the voxel-wise concordance change in left middle frontal gyrus was negatively correlated with non-perseverative errors in WCST of ADHD group. Whereas, we did not find a correlation between them in the HCs. WCST is widely used to assess executive function, including cognitive flexibility, working memory, continuous attention and response inhibition . Non-perOur findings highlighted the role of frontal lobe in the neuropathological mechanism of ADHD, and demonstrated that concordance of R-fMRI indices can serve as a biomarker to indicate the deficit in frontal lobe. To date, the diagnosis of ADHD is still based on symptom scores from clinical interviews, which can be subjective and can be influenced by many factors, such as the experience, stereotype, and cultural differences. Neuropsychiatric biomarkers can help to build a standard and objective diagnosis system, which can benefit the patients. R-fMRI is among the neuropsychiatric tools that have the potential to find the biomarkers . Such biFurther, the behavioral dysfunction may be too subtle to be measured, just like the non-perseverative errors that failed to be capture by the current study. However, as mentioned above, the R-fMRI indices as proposed by the current study are able to evaluated the maladaptive of brain functions.This study has several limitations. First, no significant differences were found among some dynamic indices, which may be due to the relatively small sample size. A larger sample size is needed to validate the alterations in dynamic brain function in ADHD and examine the relationship between dynamic variables and psychological data. Second, the current study only focus on the BOLD signal from gray matter. However, previous studies have demonstrated the white matter signals also provide valuable information ,b. HenceThe current study investigated the variability and concordance of a range of commonly used R-fMRI indices in ADHD. We found that patients with ADHD exhibited increased dALFF variability in right middle occipital gyrus and decreased dALFF variability in left middle frontal gyrus. Meanwhile, we found that voxel-wise concordance of a series of R-fMRI indices in patients with ADHD was decreased in frontal lobe, and a greater decrease was related to worse WSCT performance. These findings suggest that dynamic analysis and functional concordance may provide new insights into the neuropathological mechanism of ADHD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.FL and JT analyzed the data and wrote the manuscript. RZ analyzed the data. SC and AQ collected the MRI data. CY, XZ, and BC evaluated the subjects. ZH and MW designed and supervised the experiment, and finalized the manuscript. All authors reviewed the manuscript and made significant contribution to this manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The incidence of preterm birth and subsequent low birth weight (LBW) are vital global public health issues. It contributes to high infant and child mortality in the early stages of life and later on in adult life; it increases the risk for non-communicable diseases. The study aims to understand the socio-economic status-related inequality for LBW among children in India. It hypothesises that there is no association between the socio-economic status of the household and the newborn’s LBW in India.N = 127,141). The concentration index (CCI) and the concentration curve (CC) measured socio-economic inequality in low birth status among newborns. Wagstaff decomposition further analysed key contributors in CCI by segregating significant covariates.The study utilised data from the fourth round of the National Family Health Survey, a national representative cross-sectional survey conducted in 2015-16  and the educational status of mothers (43.4%) for LBW among children in India. Additionally, the body mass index of the women (28.4%), ante-natal care (20.8%) and residential status (− 15.7%) explained SES related inequality for LBW among children in India.Adequate attention should be given to the mother’s nutritional status. Awareness of education and usage of health services during pregnancy should be promoted. Further, there is a need to improve the coverage and awareness of the ante-natal care (ANC) program. In such cases, the role of the health workers is of utmost importance. Programs on maternal health services can be merged with maternal nutrition to bring about an overall decline in the LBW of children in India.The online version contains supplementary material available at 10.1186/s12887-021-02988-3. The incidence of preterm birth and subsequent low birth weight (LBW) are vital global public health issues . It contLBW is one of the most important indicators to measure the socio-economic development of the community. The mother’s socio-economic characteristics are directly linked to the child’s health and well-being. Studies have shown that mother’s education, knowledge and media exposure are significantly linked to reducing LBW in low and middle-income countries, including India , 5, 7. ISince the mother’s health and nutritious characteristics play a significant role in the outcomes of preterm birth and LBW among their children, it is, therefore, essential to provide proper health, diet, and nutritional care to mothers during pregnancy , 7, 16. The incidence of newborn LBW is an important predictor of newborn health, growth, cognitive development, and survival . The motIn this way, there are many predisposing risk factors that influence LBW in India. Very few studies have compared the prevalence of LBW across different gradients of women and children, such as socio-economically, demographically, and environmentally in India and across regions. A dearth of literature is found to understand the multiple determinants that are pervasive to LBW in India and across regions. Therefore, the study aims to understand the socio-economic status-related inequality for LBW among children in India. The study further hypothesises that there is no association between the socio-economic status of the household and the newborn’s LBW in India.We used data from the fourth round of the National Family Health Survey (NFHS), a nationally representative cross-sectional survey conducted in 2015–16. The International Institute for Population Sciences (IIPS) in Mumbai has conducted four rounds of NFHS surveys under the aegis of the Indian Ministry of Health and Family Welfare (MoHFW). The NFHS-4’s main goal was to offer vital statistics on health and family welfare, as well as data on emerging difficulties in these areas. As a result, the survey collects data on population health and nutrition for men, women, and children in all 36 states and union territories of India. To obtain estimates for India as a whole and its states, the NFHS-4 used a two-stage stratified sampling approach. The details can be obtained from elsewhere . With a For the newborn’s birth weight, a question was asked to the mothers ‘Was (NAME) weighed at birth? If the response was yes, further, ‘How much did (NAME) weigh? The responses were recorded as weight in kilograms from card and kilograms from recall. Among 259,627 sampled children, 57,451 children were not weighed at birth, and 8818 were “don’t know” cases. After excluding children whose birth weight data were not available, finally, we had 193,358 children with LBW data. In order to obtain better estimates for the body mass index (BMI), the study excluded women who were presently pregnant and women who had given birth in the previous 2 months. The NFHS-4 gathered data on the three youngest children from the birth history who were born within the survey’s reference period. To eliminate recall bias, we only included the most recent birth in the 5 years before to the survey. Also, for the second and third most recent births in history, information on Antenatal Care (ANC) associated characteristics was not accessible. As a result, we had a total of 127,141 index children in our analytical sample. Table All newborns weighing less than 2.5 kg (< 2500 g) at the time of birth was considered low birth weight (LBW) children. The outcome variable was dichotomous: 1 ‘yes’ if newborns weighing less than 2.5 kg at the time of birth and 0 ‘no,’ otherwise , 24–26.2, normal: 18.5 to < 24.99 kg/m2 and overweight/obesity: ≥25.0 kg/m2) [Based on the literature, we identified potential risk factors of LBW and included them as exposure variables in the analysis , 22–24. 0 kg/m2) , and ant0 kg/m2) , and rel0 kg/m2) . Further0 kg/m2) . We incl0 kg/m2) . The flu0 kg/m2) . We termThe distribution of the study population was depicted using descriptive statistics. The factors linked with the outcome variable were also identified using bivariate and multivariate logistic regression analysis. To further understand the relationship (significant or not significant) between outcome variables and predictors, a Chi-square test was used.The wealth quintile was the most important factor in determining a family’s economic state. To undertake decomposition analysis and produce the concentration index, we first calculated the wealth score (CCI). The children were then graded and divided into five equal categories, each having 20% of the children, based on their wealth ratings , 32. To The concentration index can be written as follows:yi is the outcome variable index; R is the fractional rank of individual i in the distribution of socio-economic position; μ is the mean of the outcome variable of the sample, and cov denotes the covariance [Where C is the concentration index; variance . The indThe concentration index takes a negative value if the curve is above the line of equality, suggesting a disproportionate concentration of inequality among the poor (pro-rich) , 32. If yi, the intercept α, the relative contribution of xki and the residual error εiWagstaff decomposition methodology was used to decompose the concentration index . The conεi is an error term, given the relationship between yi and xki, the CCI for y (C) can be rewritten asWhere μ is the mean of yi, xk, βk is the coefficient from a linear regression of outcome variable, Ck is the concentration index for xk .Where k. Second, a residual or ‘unexplained’ factor Here C is the outcome of two components: First, there are the determinants, or ‘explained’ factors, which are comparable to the weighted accumulation of the regressor’s concentration indices, where one unit change in the outcome variable corresponds to one unit change in the explanatory variable , 32. Thec groups . The perc groups , 32. Thec groups .The percentage distribution of the study population by background characteristics is presented in Table The percentage distribution of LBW among children by background characteristics in India is presented in Table Table Figure Figure Estimates of decomposition analysis for the contribution of various explanatory variables for LBW children in India is presented in Table In the present paper, we have tried to identify the determinants of a child’s LBW by analyzing the fourth round of the National Family Health Survey. We found that LBW of a child was due to the following reasons: (i) Mother’s individual characteristics such as her age (below 19 years), low BMI status, with no ANC services and education; (2) Mother’s household characteristic such as if she belonged to the poorest wealth quintile, and scheduled caste; (3) If the child was a female and from a higher birth order & interval. We also found that the LBW of the child was concentrated in the poorest sections of society. Further, the mother’s wealth quintile, region and educational status contributed to the maximum while explaining the socio-economic related inequality for LBW among the children in India.The present study results indicate that women who had low BMI status were more likely to have children with LBW status. This observation is in accordance with a study that shows that girls with low birth weight are likely to give birth to LBW infants . FurtherOur study found that female infants had a higher likelihood of being born LBW. Though the study finding is similar to few studies , 49, it The socio-economic inequality in LBW newborns in India was measured by Concentration Index and Concentration curve. The analysis showed that LBW is mostly concentrated in the North-eastern regions of the country, followed by the South and Northern parts of the country. However, a study shows that the Northern states of India had the highest concentration of LBW children . LiterarThe present study uses a nationally representative recently published sample of a well-known large-scale survey in India. Therefore, the study results can be generalised well. However, we acknowledge some limitations too. First, because of the cross-sectional nature of the data, we could not draw any causal relationship between the variables. Second, another limitation of the cross-sectional household survey is that birth weight is estimated from the records of the newborn in health facilities. However, the birth weight of those born at home are not available.Since India contributes to the highest infant LBW in the southern region, identifying the risk factors of LBW has significant policy implications. Adequate attention should be given to the mother’s nutritional status; timely intake of iron and folic acid tablets during pregnancy can be a way of achieving it. Awareness of health education and usage of health services during pregnancy are few other important things that can be taken up at the household and community level. Further, there is a need to improve the coverage and awareness of the ANC program. Hence, the role of the health workers is of utmost importance. Programs on maternal health services can be merged with maternal nutrition to bring about an overall decline in the LBW of children in India.Additional file 1: Table S1. STROBE Flow Diagram.


We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn7-like transposons, such as insertion sequences and toxin-antitoxin modules, and of genes involved in recombination, anti-MGE defense, and antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty-two Tn7-like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn7-like transposons, defines their evolutionary relationships, and provides a large-scale functional classification of prokaryotic genes mobilized by transposition.Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn Horizontal gene transfer (HGT) between prokaryotic genomes is one of the principal forces shaping prokaryotic genome evolution –6. The m–7–10–16–7 from Escherichia coli. Detailed biochemical dissection of Tn7 identified five “core” genes that collectively execute transposon excision, target site selection, and integration  are the most abundant class . At a miegration , 23. Thecleavage . A seconcleavage . Transpocleavage , 27. Thecleavage .7-like transposons recognize their target sites via a CRISPR spacer-guided mechanism  in a given locus  for the core genes of the E. coli Tn7 (tnsABCDE) and known Tn7-like transposons were used to identify homologs in the database of prokaryotic genomes. Loci were considered candidate Tn7-like transposons if they encompassed adjacent open reading frames (ORFs) with significant sequence similarity to the core transposase TnsB and at least one other Tn7 HMM. The TnsB transposase sequences were employed for phylogenetic reconstruction, given that TnsB is required for transposition, whereas the other subunit of the heteromeric transposase, TnsA, is dispensable , Verrucomicrobia (5), Acidobacteria (4), Fibrobacteres (4), Aquificae (3), Spirochaetes (2), Deferribacteres (2) and Planctomycetes (1) , and the leaves from each clade were assigned a taxonomic host phylum using an 80% consensus rule. The first clade includes TnI-B CAST , and oth53 , and the representative sequence is provided for each transposon (“Tn_rep” column). The community to which each transposon belongs is listed for each hierarchical level. Download Table S1, XLSX file, 4.9 MB.(A) Metadata for all TnsB transposases. For each TnsB protein, the host taxonomy and nucleotide coordinates are recorded. The leaf ID corresponds to the label of the Newick tree. The “motif ID” column contains the MEME-formatted consensus sequence of the predicted TnsB binding site. Incomplete transposons whose ends could not be predicted do not have a motif. (B) Comparison of predicted versus known transposon boundaries. The distance (in base pairs) between the outermost predicted TnsB binding site and the previously determined transposon boundary is reported. The boundaries of two transposons could not be predicted automatically (N/D). (C) Metadata for all complete transposons. The nucleotide coordinates and host DNA classification  are reported for all complete TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7, Tn5090, Tn5053, and Tn552 possess arrays of inverted repeats located subterminal to the 5′ and 3′ boundaries of the transposon . Under the premise that phylogenetically close transposases would utilize conserved binding site sequences, any discovered arrays of inverted repeats flanking a given transposase were compared to those found for another, closely related homolog, if available. If the inverted repeat sequences were conserved among ≥50% of close transposase homologs and satisfied additional criteria , the arrays were predicted to be the LEs/REs of the respective transposons. The computationally predicted TnsB binding sites were consistent with biochemical data on Tn7 transposition, where three TnsB binding sites were present at the left end and four at the right end , which was present in 73 to 97% of the transposons in each clade. The PDEXK-family transposase tnsA is nearly ubiquitous in clade 1 (92%) but relatively uncommon in clade 3 (28%); this pattern is inverted for the tyrosine and serine superfamily resolvases (tnsR) . Only the transposons in clades 1 and 3 contained a homolog of tnsE, which promotes the integration of Tn7 into replication forks, including those on plasmids , resulting in a final set of 6,549 putatively complete Tnnsposons . Besidesy ATPase . The nex clade 3 %; this p homolog . This dito 29 kb , but mucto 29 kb . The carProteobacteria, and it could be divided into 15 subcommunities  and multiple classes of Proteobacteria, with an observable contribution of plasmid-mediated transfer  that are smaller, on average (B). A resolution of 140 yields the highest mean community persistence and was therefore selected to construct the final network (C). Construction of smaller communities marginally decreases the phylogenetic level of the communities’ lowest common ancestor, operationally defined as the consensus host of ≥80% of the transposons in a community (D). Download Copyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bacillus cereus transposons from clades 1 and 2 showed minimal sequence similarity between their core proteins but carry nearly identical arsenate resistance operons and because of that belong to the same community  are compared, showing that both mobilize a predicted arsenate resistance operon (A). Conversely, the cargo carried by two transposons from the same clade (clade 3) mobilize different cargo repertoires, even though both are integrated in the same genome (B). Download FIG S2, EPS file, 0.8 MB.Closely related TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7-like transposons found to be integrated into completely sequenced bacterial genomes (n = 739) were compared against randomly selected loci of equal lengths. All ORFs were annotated against the Clusters of Orthologous Groups (COG) database , particularly those of the IS3 family, that are present in ∼30% of the transposons . Examples of two colistin-resistance genes that are part of a putative “nested” transposon (B). Two IS21-family transposons  flank nitrogen-cycling genes (purple) (C). Unclassified, serine resolvase-carrying transposons are the cargo of multiple Tn7-like transposons (D). Download FIG S3, EPS file, 2.3 MB.The cargo of TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The next most highly enriched set of genes, apart from genes of unknown function, comes from the defense COG class. A search of the cargo for genes involved in biological conflicts uncovered numerous, diverse innate immune systems, the most common and widespread being restriction-modification modules . The nex66–10.1128/mBio.02938-21.4FIG S47-like transposons. Download FIG S4, EPS file, 2.1 MB.Innate immune systems mobilized by transposons. Selected examples of innate immune systems (purple) carried as cargo by TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7-like transposons mobilize a variety of toxin effectors, including PIN family ribonucleases, RelA/SpoT-like nucleotidyltransferases, ParE-family mRNA interferases, ADP-ribosyltransferases, and HipA-family kinases (7-like transposons.Mobile genetic elements often encompass toxin-antitoxin (TA) gene pairs, which frequently colocalize with innate immune systems , 73 and 74– kinases . Several kinases , implica kinases . Thus, T10.1128/mBio.02938-21.5FIG S57-like transposons. Download FIG S5, EPS file, 1.2 MB.Toxin-antitoxin and abortive infection systems mobilized by transposons. Selected examples of toxins (purple) and antitoxins  carried as cargo in TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cas genes. In this survey, 410 transposons were shown to harbor at least one cas gene or a CRISPR array; of these, all but 14 belong to one of three subclades in the TnsB phylogenetic tree . Examples 2, 4, and 5 carry an interference or adaptation module, whereas the most of the remaining transposons possess only stand-alone components of CRISPR-Cas (not shown). Download FIG S6, EPS file, 1.0 MB.Capture of CRISPR-Cas systems by TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02938-21.7FIG S7FIG S7, EPS file, 1.4 MB.Replicative transposition is predicted by the absence of TnsA and presence of TnsR. The presence of core proteins in ≥50% of the transposons represented by each leaf are indicated on the outer rings of the TnsB tree. The resolvases of clades 2 and 3 are highlighted in purple, which do not co-occur with TnsA. Download Copyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7-like transposon cargo is “replication, recombination, and repair” and, more specifically, genes encoding enzymes of the tyrosine and serine integrase/resolvase superfamilies. Enzymes from both superfamilies are encoded by diverse MGEs and catalyze site-specific rearrangements in DNA that are involved in transposon relocation from a donor to an acceptor site  is responsible for the apparent commonality of the glycine cleavage pathway. To explore the larger pool of antibiotic resistance genes, which belong to various COG categories, the cargo was searched against a database of genes with experimentally demonstrated roles in antibiotic and xenobiotic resistance  or stress-resistance genes (B). Note the log scale. Download FIG S8, EPS file, 1.4 MB.Antibiotic and stress resistance genes mobilized by TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7 and other families of transposons because of their role in aggregating antibiotic resistance genes . The cassettes are dominated by qacE (334 cassettes) and dfrA1 (157 cassettes), which confer resistance to ammonium-based disinfectants and trimethoprim, respectively. Integrons carrying homologs of genes that provide resistance to aminoglycosides, beta-lactams, phenicols, sulfonamides, macrolides, lincosamides, quinolones, and bleomycins were also captured by Tn7-like transposons, illuminating the role of integrons in expanding the diversity of the antibiotic and stress resistance cargo.To elucidate how antibiotic and stress-resistance genes are captured by transposons, the cargo was examined for the presence of integrons. Integrons are MGEs that site-specifically incorporate “cassettes” of DNA and constitute a notable component of the cargo of Tnce genes , 84. Alt7-like transposons to mobilize genes that modulate cellular metabolism.The transposons mobilizing genes involved in biosynthetic pathways and central carbon metabolism were analyzed next. The most common biosynthetic pathways in transposons are those for fatty acids and menaquinones, including COG1028 , COG1960 (acyl coenzyme A [acyl-CoA] dehydrogenase), COG0183 (acetyl-CoA acetyltransferase), and COG0596 (MenH-family esterase). These enzymes appear in a variety of genetic contexts, making their specific roles and end products difficult to infer, but generally are involved in the synthesis or degradation of “auxiliary” compounds. Additionally, multiple transposons carry genes involved in central metabolic processes, including glycolysis, gluconeogenesis, the pentose phosphate pathway, and the tricarboxylic acid cycle . The gen10.1128/mBio.02938-21.9FIG S97-like transposons. Central carbon metabolism genes are highlighted in purple, with their respective pathway labeled above each transposon. Abbreviations: Mdh, malate/lactate dehydrogenase (COG0039); OxdD, oxalate decarboxylase (COG2140); Eda, 2-keto-3-deoxy-6-phosphogluconate aldolase (COG0800); LeuB, isocitrate/isopropylmalate dehydrogenase (COG0473), PckA, phosphoenolpyruvate carboxykinase (COG1866); Tdh, threonine dehydrogenase (COG1063). Download FIG S9, EPS file, 1.8 MB.Central carbon metabolism genes mobilized by TnCopyright © 2021 Benler et al.2021Benler et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 7 proteins involved in transposition recovered thousands of predicted transposons in diverse bacterial phyla, underscoring the mobility of these elements. The phylogeny of TnsB demonstrates the relationship between Tn7 and other well-characterized transposons, including Tn5053 and the recently described CASTs. Besides the phylogenetic coherence of the transposase, all these transposons possess similar architectures of inverted repeats at their left and right ends that mark the boundaries of the transposon. Moreover, within these boundaries, most encode a transposition-regulating ATPase (tnsC) and a target site-selecting protein (tnsD) flanking the core transposase. These shared features are the hallmarks of a vast, distinct group of MGE informally dubbed Tn7-like transposons  that most likely mediates replicative transposition. The target site-selecting gene tnsE is absent in most clades of transposons, although the actual spread of this gene might be underestimated due to the weak sequence conservation. Similarly, the CRISPR-Cas target site-selecting systems are relatively rare, but additional CASTs might be discovered with improvements in transposon delineation. The evolutionary gain and loss of tnsAER and the specialized CRISPR-Cas systems illustrate the modularity of the transposition machinery of the Tn7-like transposons.Tnposition , 86. Multogether , evincin7-like transposons exhibit an even greater plasticity. To survey the biological functions of the cargo, a comparison against genes located outside the transposons was undertaken. An unexpected outcome of this analysis was that the most highly enriched function, the mobilome, was not explained by the presence of core Tn7 proteins alone but also by substantial contributions from other MGEs, specifically, IS elements. The enrichment of ISs suggests a stepwise process of gene capture by Tn7-like transposons, beginning with the formation of a compound transposon by two ISs at a locus outside a Tn7-like element, followed by the compound transposon jumping into the boundaries of the element, yielding a nested transposon. Such nesting of MGEs has been previously observed in individual transposons and other MGEs  were collected from the Pfam database using the following accession numbers: TnsA, PF08721 and PF08722; TnsB, PF00665; TnsC, PF11426 and PF05621; TnsD, PF15978; TniQ, PF06527; TnsE, PF18623. Additional MSAs of Tn7-like core proteins were generated from CASTs, and all MSAs were converted to HMMs with HMMer (v. 3.1b2). For the purpose of a comprehensive search for Tn7-like transposons, a nucleotide database encompassing the NCBI whole-genome shotgun (WGS) database and the MG-RAST database of metagenomes  a hit to the TnsB HMM with a bitscore of ≥60, (ii) a hit to at least one other Tn7 HMM, and (iii) two hits in a putative operon, which is operationally defined as two codirected ORFs separated by less than 50 bp of noncoding sequence. All loci satisfying these criteria were considered candidate Tn7-like transposons, and the putative TnsB orthologs were subjected to phylogenetic analysis.Multiple-sequence alignments (MSAs) of the five core genes from agenomes  was prepagenomes  for ORF 7-like transposon was extracted from the NCBI taxonomy database using the Entrez suite of command line tools. If a sequence was not indexed in the database, it was assigned to the domain Bacteria, given that Tn7-like transposons have not so far been identified in genomes of archaea or viruses (see Results). Contigs were scanned using ViralVerify . The same filtering approach for the BLASTn output described above was applied to the FIMO output, with the following additional criteria: (i) the spacing between any two instances of a motif must be ≤75 bp, (ii) the total length of all motif instances on a sequence must be ≤120 bp, (iii) the motif cannot be present on 5 or more intergenic sequences, and (iv) the product of the motif’s estimated false-discovery rate  must be less than 0.01. Criteria 1 and 2 were enforced to match the spacing and combined length of TnsB binding sites in Tn7 . Each file was input individually to TomTom  with HHblits   and clus HHblits , enforcicas genes, representative ORFs were searched using PSI-BLAST against a database of multiple sequence alignments obtained from a recent survey   with deft survey . For inst survey .https://github.com/ctSkennerton/minced) with the default settings. Integron attC sites were annotated using IntegronFinder (v. 2.0)   against (v. 2.0) . Annotat(v. 2.0) .The nucleotide sequences of the transposons were dereplicated at 99% average nucleotide identity across 95% of the contig length using dRep  and assoThe enrichment or depletion of COG functional categories for the subset of transposons in completely sequenced bacterial genomes were calculated as described previously . BrieflyThe COG annotations of transposon-borne ORFs were extracted and assigned to their respective COG pathways. The completeness of the pathway was calculated as the number of COGs from the pathway represented in the transposon divided by the total number of COGs constituting the pathway. Pathways were discarded if represented in fewer than 10 transposons.https://ftp.ncbi.nih.gov/pub/yutinn/benler_2021/Tn7/source_data/. The code for delineating Tn7-like transposon boundaries is publicly available at https://github.com/sean-bam/Tnacity.All sequences are publicly available from the NCBI and MG-RAST databases. The nucleotide coordinates of all transposons are provided in


Bifidobacterium bifidum (Bf-688) can improve clinical characteristics and gut microbiomes among patients with attention-deficit/hyperactivity disorder (ADHD). This open-label, single-arm trial consisted of 30 children aged 4–16 years who met the criteria for ADHD diagnosis. Each subject took Bf-688, with one sachet in the morning and one in the evening (daily bacteria count 5 × 109 CFUs), for 8 weeks. Patients’ clinical symptoms were assessed using the Swanson, Nolan, and Pelham Rating Scale (SNAP-IV). We collected stool samples at the baseline, the 8th week, and the 12th week for gut microbiota examination. During the 8-week Bf-688 supplement period, patients’ inattention symptoms and hyperactivity/impulsive symptoms improved, and their weights and BMIs increased. For gut microbiota, the Firmicutes to Bacteroidetes ratio (F/B ratio) decreased significantly. LEfSe analysis revealed that Firmicutes significantly decreased while Proteobacteria significantly increased during the 8-week treatment period. After Bf-688 was discontinued for 4 weeks (12 weeks from baseline), Bacteroidota significantly decreased and Shigella significantly increased. The probiotic Bf-688 supplement was associated with an improvement of clinical symptoms and with weight gain among ADHD children. Furthermore, gut microbiota composition was significantly altered by the Bf-688 supplement. A future randomized control trial is warranted to verify these findings.This study aimed to examine whether probiotics supplements using  Attention-deficit/hyperactivity disorder (ADHD) is one of the most common mental disorders that occur in childhood. It affects approximately 3 to 10% school-age children around the world . ADHD maLactobacillus rhamnosus, LGG) during the first six months of life can reduce the risk of ADHD and autism spectrum disorders (ASD) in later childhood [Lactobacillus plantarum PS128 improved inattentive/hyperactive symptoms in children with ASD [Bifidobacterium abundance and had potential implications for modulating and regulating ADHD behavior [One prospective study showed that supplementing a child’s diet with probiotics  species constitutes one of the dominant taxa amongst Bifidobacterial communities [B. bifidum has been demonstrated to improve the quality of life in individuals with rhino-conjunctivitis seasonal allergies [B. bifidum TMC3115 strain (TMC3115) may exhibit beneficial effects on the serum cholesterol metabolism of subjects with dyslipidaemia via the modulation of their intestinal microbiota [B. bifidum MIMBb75 (SYN-HI-001) has been shown to substantially alleviate the symptoms of irritable bowel syndrome [B. bifidum G9-1 (BBG9-1) improves dysbiosis, which results in an increase in organic acids and improved neurotransmission (including dopamine) [Lactobacillus species and B. bifidum could improve rotational behavior, cognitive function, and neuronal damage in patients with Parkinson’s disease (PD) [A previous case-control study revealed a nominal increase in ynthesis . The Bifmunities . B. bifillergies , and it llergies . Anothercrobiota . Furthersyndrome . B. bifiopamine) . In addiase (PD) .B. bifidum Bf-688 strain has not yet been investigated in children with ADHD. We hypothesized that altering the dysbiosis of gut microbiota may be associated with improvements in ADHD clinical symptoms. This open-label, single-arm clinical study aimed to examine whether Bf-688 supplementation could improve the clinical symptoms of ADHD and facilitate healthy weight gain. We also investigated whether 8-week Bf-688 supplementation would change gut microbiome composition and whether any changed composition would last for more than four weeks after discontinuing Bf-688.Numerous studies have shown that probiotic supplementation can have a positive effect on the course of neurodevelopmental disorders, including ADHD . HoweverThis research protocol was approved by the Institutional Review Board (IRB) at Chang Gung Memorial Hospital in Taiwan. Eligible patients with ADHD were recruited from the outpatient Department of Child Psychiatry at Chang Gung Children’s Hospital in Taiwan. We explained the protocols of this study to participants and their parents or guardians prior to their entry into this study; written informed consent was obtained from both the children and their parents/guardians upon their agreement.The inclusion criteria for patients with ADHD were as follows: (a) a clinical diagnosis of ADHD made by a senior child psychiatrist via structured interviews using the Diagnostic and Statistical Manual of Mental Disorders (DSM–5) ,29; (b) 9 CFUs). At the baseline, 2nd, 4th, and 8th weeks for each subject, ADHD symptoms were assessed using the Swanson, Nolan, and Pelham Rating Scale (SNAP-IV) parent-form [This open-label, single-arm trial recruited 30 children aged 4–16 years who met the criteria of ADHD diagnosis. Each subject took Bf-688 for 8 weeks, with one sachet in the morning and one in the evening . DNA concentration was determined by a Qubit 4.0 Fluorometer  and adjusted to 1 ng/ul for the process described below.The full-length 16S gene sequence (V1–V9 regions) was amplified by barcoded 16S gene-specific primers. According to the amplification of the full-length 16S gene with barcoded primers for multiplexed SMRTbell library preparation and sequencing procedure (Pacbio), each primer was designed to contain a 5′ buffer sequence (GCATC) with a 5′ phosphate modification, a 16-base barcode, and the degenerate 16S gene-specific forward or reverse primer sequences . The degenerate base identities were as follows: R = A, G; Y = C, T; and M = A, C. In brief, 2 ng of gDNA was used for the PCR reaction, which was carried out with KAPA HiFi HotStart ReadyMix (Roche) under the following PCR conditions: 95 °C for 3 min; 20~27 cycles (sample dependences) of: 95 °C for 30 s; 57 °C for 30 s; 72 °C for 60 s; and 72 °C for 5 min then held at 4 °C. The PCR products were monitored on a 1% agarose gel. Samples with a bright main strip of around 1500 bp were chosen and purified using AMPure PB beads for the library preparation procedure described below.The SMRTbell library was prepared according to the amplification of full-length 16S gene with barcoded primers for multiplexed SMRTbell library preparation and sequencing procedure (Pacbio). In brief, an equal molar of each barcoded PCR product was pooled and then 500–1000 ng of the pooled amplicon sample was used for DNA damage repair followed by End Repair/A-tailing and ligation steps in order to introduce universal hairpin adapters onto the double-stranded DNA fragments. After purification with AMPure PB beads to remove adapter dimers, the SMRTbell library was incubated with sequencing primer v4 and sequel II Binding Kit 2.1 for primer annealing and polymerase binding. Finally, we performed sequencing using the circular consensus sequence (CCS) mode on a PacBio Sequel Iie instrument to generate HiFi reads with predicted accuracy  = 30.The circular consensus sequence (CCS) reads were determined using a minimum predicted accuracy of 0.9 and a minimum number of 3 passes in the official workflow of PacBio via the SMRT Link software. After demultiplexing, the CCS reads were further processed with DADA2 in order to obtain amplicons with single-nucleotide resolutions ,32. The Alpha diversity indicates species complexity within individual samples based on seven different criteria outputs, including the following: observed-species; Menhinick’s Richness; Margalef’s richness; Shannon index; Simpson index; PD whole tree; and good coverage. Observed-species is the number of different species represented in the microbial community. Community richness was assessed using both Menhinick’s richness and Margalef’s richness, and the relative abundance and evenness accounting for diversity were evaluated by Shannon and Simpson indices. We constructed a rarefaction curve using random selection of a certain amount of sequencing data from each sample to represent the number of observed species .t-test was performed using STAMP software [For statistical analysis, significance of all species among groups at various taxonomic levels was detected using differential abundance analysis with a zero-inflated Gaussian (ZIG) log-normal model as implemented in the “fitFeatureModel” function of the Bioconductor metagenomeSeq package . Furthersoftware . Statistsoftware . In brieIn total, 30 children with ADHD  were recruited for this study . In our p < 0.05). During the eight-week period of ADHD patients receiving oral probiotic Bf-688, patients’ body weights , a relevant microbial biomarker of ADHD, significantly decreased from baseline to week 8 (p = 0.0012) (p = 0.0026). 0.0012) . The F/BBacteroidota, Firmicutes, and Proteobacteria, respectively. Bacteroides vulgatus, Bacteroides steroids, Bacteroides uniformis, Bacteroides fragilis, and Shigella flexneri were the five most abundant species among patients.Firmicutes significantly decreased while Proteobacteria significantly increased. Our previous study [Bacteroides ovatus (B. ovatus) in ADHD subjects. In the current study, an 8-week probiotic (Bf-688) supplement reduced the abundance of B. ovatus in ADHD subjects. Bacteroidota significantly decreased while Shigella significantly increased.We further analyzed the gut microbiota composition of ADHD subjects. The Shannon index and Simpson index were analyzed in order to determine species richness; we found no significant differences between the baseline, week 8, and week 12 . Figure us study  found a Collinsella aerofaciens and Erysipelatoclostridium ramosum were collectively abundant species. However, Clostridium viride showed relatively high abundance in the samples at baseline (week 0). The abundances of Granulicatella elegans and Blautia hydrogenotrophica were relatively high at week 0 and decreased at week 8; then, they were re-elevated at week 12.The relative abundances of dominant species are shown in B. bifidum (Bf-688 strain) as a supplement for ADHD, and ADHD symptoms improved during the 8-week test period. Various strains of B. bifidum have been demonstrated to improve several physical conditions, including rhinoconjunctivitis-specific quality of life [B. bifidum G9-1 (BBG9-1) potentially improves dysbiosis, which results in modulations of organic acids and neurotransmission (including dopamine) [This study is the first one to use  of life , reducin of life , and imp of life , irritab of life , and Par of life . B. bifiopamine) . Furtheropamine) . We propBifidobacterium species have been proven to reduce the incidence of necrotizing enterocolitis in preterm infants [B. bifidum HI-MIMBb75 was rated as very good or good by 91% of participants [Bifidobacterium may have potential beneficial effects on gastrointestinal symptoms in ADHD patients. Whether supplements of Bf-688 improve ADHD children’s appetite or weight gain requires further validation with a randomized control trial.Our clinical study revealed that ADHD patients’ body weights and BMIs significantly increased after Bf-688 administration.  infants . A previicipants . ADHD meicipants . The resFirmicutes to Bacteroidetes ratio (F/B ratio), a microbial biomarker, significantly decreased from baseline to week 8 and then re-elevated from week 8 to week 12. The F/B ratio evolves during different life stages [B. bifidum TMC3115 supplement reduced allergic scores, improved anti-inflammatory responses, and reduced the serum levels of IgE in infants. In contrast, a B. bifidum supplement increased the proportion of probiotics and reduced the proportion of pathogens [B. bifidum, maintained CD4+ lymphocytes and produced a less inflammatory cytokine profile, possibly as a result of changes in their microbial communities; furthermore, this profile more closely resembled those reported in healthy younger populations [Bacteroides and Sutterella may be potential gut microbiota markers of ADHD and that dietary content may be associated with gut microbiome composition [B. bifidum supplements changed gut microbial composition (reduced F/B ratio); however, the changed composition diminished after discontinuing B. bifidum supplements.Bacterial taxonomic distributions changed during the administration of Bf-688 supplements in ADHD patients. The e stages , and a he stages . A previathogens . Anotherulations . Our casposition . Taken tOur study has certain limitations. First, this study design was an open-label, single-arm study rather than a randomized control trial. The current study lacked a comparison group, and ADHD clinical symptoms were subjectively determined. The therapeutic effect of Bf-688 could have been easily influenced by the placebo effect. Second, the sample size was small. Only 23 patients completed the 8-week study period; thus, statistical power was limited. In addition, the age range of the participants was wide (4–16 years), and changes of gut microbiomes and behavioral patterns may be different between age groups. However, subgroup analyses stratified by age were not performed in this study as a result of the small sample size. Third, dietary habits or lifestyles may influence the gut microbiome. Although we asked patients not to change their dietary patterns and antibiotic and anti-inflammatory drugs were prohibited, the gut microbiota may still have been altered by other environmental factors. Fourth, cytokine or immunological function levels were not measured in this study. Therefore, the manner probiotics interact with gut microbiota and behavioral symptoms still requires further investigation. Additional studies are needed to clarify the molecular mechanisms that underly the gut–brain axis as the basis for modifying commensal microbiota or their functions for targeting ADHD .Bf-688 improved clinical symptoms of ADHD and increased body weights and BMIs of ADHD children. Furthermore, gut microbiota composition was significantly altered by the Bf-688 supplement. A future randomized control trial is warranted to verify the treatment effects of probiotics (Bf-688) on ADHD symptoms.The results in this study suggest oral probiotic