Agathobacter, Anaerostipes, and Lachnospiraceae. Top differentially abundant bacteria and representative biological pathways were identified in children with ADHD using linear discriminant analysis (LDA) effect size (LEfSe), and the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, respectively. The plasma levels of TNF-α were significantly lower in children with ADHD than in HCs. Within the ADHD group, the levels of TNF-α were negatively correlated with ADHD symptoms and diversity of the gut microbiome. Our study provides new insights into the association between gut microbiome dysbiosis and immune dysregulation, which may contribute to the pathophysiology of ADHD.Attention-deficit/hyperactivity disorder (ADHD) is a common childhood mental disorder with undetermined pathophysiological mechanisms. The gut microbiota and immunological dysfunction may influence brain functions and social behaviours. In the current study, we aimed to explore the correlation of gut microbiome imbalance and inflammation in the pathophysiology of ADHD. Forty-one children with ADHD and thirty-nine healthy-control (HC) individuals were recruited. Faecal samples from all participants were collected and submitted for 16 S rRNA V3–V4 amplicon microbiome sequencing analysis. The plasma levels of 10 cytokines, including TNF-α, IL-6, IL-1β, IL-2, IL-10, IL-13, IL-17A, IFN-α2, IFN-γ, and MCP-1, were determined using a custom-made sandwich enzyme-linked immunosorbent assay (ELISA) developed by Luminex Flowmetrix. There was no significant difference between the ADHD and HC groups in species diversity in the faeces, as determined with α-diversity and β-diversity analysis. In the ADHD group, three differentially abundant taxonomic clades at the genus level were observed, namely  Children with attention-deficit/hyperactivity disorder (ADHD) suffer from failures in academic performance and interpersonal relationships, and from multiple psychiatric comorbidities, which, without proper interventions, may persist into adulthood . AlthougDesulfovibrio, order Clostridiales [Bacteroides and Sutterella was different between children with ADHD and HCs [Bifidobacterium, which is involved in the dopamine neural reward system, suggested that Bifidobacterium abundance may serve as a biomarker for ADHD [Faecalibacterium, a genus of the Ruminococcaceae family, Anaerotaenia and Gracilibacter at the genus levels have been associated with attention deficit, which indicates that dysbiosis in gut microbiota is potentially involved in the pathophysiology of ADHD [To analyse the phylogeny and taxonomy of samples with complex microbiomes or from complex environments, 16 S ribosomal RNA (rRNA) sequencing is commonly used for amplicon sequencing . Althougridiales . Our resfor ADHD , 18. Mor of ADHD –21. BaseThe intestinal microbiota may influence the development of multiple organs, including the brain, lungs and the immune system and overall body growth , 23. In Imbalanced cytokine levels may represent an important linking mechanism between ADHD and allergies , 31. ForWe hypothesised that dysbiosis of gut microbiota may be partly associated with the pathogenesis of ADHD. In addition, we proposed that immunological dysregulation is involved in alterations of the gut microbiome. Therefore, our study aimed to explore the correlation between gut microbiome imbalance and inflammatory cytokine levels in the pathophysiology of ADHD.Our study protocol was approved by the Institutional Review Board (IRB) at Chang Gung Memorial Hospital in Taiwan (IRB No. 201702019A3). Children with ADHD were recruited from Chang Gung Children’s Hospital in Taiwan; HCs were recruited from the community close to the hospital. Study protocols were explained to the participants and their parents or guardians prior to their entry into this study, and informed consents were signed by both the participants and their caregivers upon their agreement.The following inclusion criteria for patients with ADHD were applied: (a) children diagnosed with ADHD, as confirmed by a senior child psychiatrist through structured interviews using the Kiddie-Schedule for Affective Disorder and Schizophrenia-Epidemiological Version (K-SADS-E) , 42; (b)The HC group consisted of children without ADHD diagnosis (aged between 6 and 16 years) within the same catchment area. HCs were also interviewed using the K-SADS-E and were not diagnosed with the aforementioned major neuropsychiatric diseases . Individuals on a vegetarian diet, suffering with major physical illnesses, or receiving any anti-inflammatory drugs, antibiotics or probiotics during recruitment were also excluded.Both, children with ADHD and HCs, were asked to collect their faecal samples by scooping a pea-sized piece of faeces in a sterile plastic tube. Samples were stored at 4 °C after collection and transferred to a −80 °C refrigerator in our laboratory within 24 h. Total genomic DNA (gDNA) was extracted from each sample using the QIAamp PowerFecal DNA Kit from Qiagen. The concentration of gDNA was determined with Qubit fluorometric quantitation prior to polymerase-chain reaction (PCR).The 16S V3–V4 region was amplified by a specific primer pair . First, faecal gDNA (12.5 ng) was used for V3–V4 region amplification. The KAPA HiFi HotStart Ready Mix (Roche) was used under the following PCR conditions: 95 °C for 3 min; 25 cycles of 95 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec; 72 °C for 5 min. The length of the PCR product was approximately 500 bp. The main fragments were then purified with AMPure XP beads, followed by a second PCR with dual indices and sequencing adapters . Finally, equal amounts of the indexed PCR product were denatured and pooled, and then the 16S library was sequenced on an Illumina MiSeq sequencer (paired-end 2 ×300 bp reads).Blood samples were obtained between 7:00 and 8:00 AM. A multiplex bead-based assay was developed using the Luminex Flowmetrix system  to investigate the cytokine levels in the plasma. Each capture antibody for an individual cytokine was coupled to a different bead set . Recombinant cytokines pre-diluted in pooled blank plasma from HCs were used as standards; test sera from patients or controls were examined with multiplex assays as previously reported , 43–45 fA senior psychiatrist confirmed the clinical diagnosis of ADHD and neuropsychiatric disorders in the enroled children with ADHD and HCs through face-to-face interviews . K-SADS-p = 0.05. The Statistical Package for the Social Sciences Version 16.0  was used for data analysis. Mean ± standard deviation or frequency were presented as variables. The chi-square test was used to analyse the sex distribution between children with ADHD and HCs. The independent t-test was used to determine the differences in demographics, clinical symptoms and neuropsychological function between patients and controls. Two-tailed p < 0.05 was regarded as statistically significant.The sample size was estimated using the software package G-Power 3.1; based on the settings of 80% power, Q < 20) were removed using the QIIME pipeline. Chimera sequences were identified using UCHIME v4.2 (with the reference Gold database) [p-values were calculated as 0.05  divided by the number of parameters in each figure.For the analysis of 16 S rRNA sequencing data, the main workflow was performed using QIIME (v1.9.1) , which catabase)  to obtaiatabase)  functionatabase)  with 97%atabase) . Ideallyatabase) . The RDPatabase)  was adapatabase) , 58 for atabase) . Moreoveatabase) . DiffereFaecal samples from 41 children with ADHD  and 39 HC individuals  were collected. The characteristics of the ADHD and HC groups in our study are summarised in Table The 16S V3–V4 library from faecal samples was constructed and sequenced in the Illumina sequencer platform according to standard procedures. We used a rarefaction curve to estimate gut microbiome species diversity. Rarefied OTU tables were generated and α-diversity metrics were established for each rarefied OTU table. For rarefaction curve generation, a certain amount of sequencing data was randomly selected from the samples to represent the number of species. When the curve became flat, a reasonable sequencing depth was acquired, indicating that more sequencing depth was likely to yield only a few additional species. Based on the chart, both the ADHD and HC samples had sufficient sequencing depth for species identification. The X axis illustrates the number of sequences (per sample), and the Y axis demonstrates the number of observed species  showed a slightly increasing trend in children with ADHD compared with HCs  algorithm to identify metagenomic biomarkers in children with ADHD. LEfSe analysis revealed differentially abundant bacterial taxa between the ADHD and HC groups Fig. . Our resnts Fig. A, B. Figr = −0.348, p = 0.028) and Shannon index   and Shannon index . Moreover, TNF-α levels in children with ADHD were negatively correlated with attention deficit  and hyperactivity/impulsivity symptoms .The plasma levels of TNF-α were significantly lower in the ADHD group than in the HC group Fig. . Except 04) Fig. . The plaα-diversity or relative abundance of Fusobacterium). Nonetheless, our data showed that a higher abundance of three genera Agathobacter, Anaerostipes and Lachnospiraceae UCG-010 was found in the ADHD group compared with the HC group. Furthermore, our study revealed a slightly higher F/B ratio in children with ADHD than in HCs. The F/B ratio has been widely investigated in previous studies regarding gut microbiota in both humans and mice and has been associated with obesity and other diseases [In this study, the 16 S rRNA sequencing platform was implemented for the screening of bacterial communities from faecal samples of children with ADHD and HC participants. Species richness in the faeces, which was determined by α-diversity and β-diversity analysis, was not significantly different between the participants of ADHD and HC groups. Our results are in line with a previous case–control study  that alsdiseases .A previous animal study showed that administration of an anti-mouse IL-6 receptor antibody (MR16-1) significantly improved the decreased F/B ratio and normalised the gut microbiota composition in susceptible mice under social-defeat stress . AnotherAgathobacter, Anaerostipes and Lachnospiraceae UCG-010. Agathobacter is a bacterial genus from the family Lachnospiraceae. The relative abundance of Agathobacter has been associated with physician-diagnosed inhalant allergies in school-age children [Anaerostipes is a Gram-positive anaerobic bacterial genus from the family Lachnospiraceae. Products of bacterial metabolism by Anaerostipes and Lachnospiraceae have been related to the aetiology of depression [Bacteroides uniformis was increased in ADHD  that may impact the initial colonisation and development of microbiota were not available in this study , 73. SecAgathobacter, Anaerostipes and Lachnospiraceae UCG-010) were observed in children with ADHD. The plasma levels of TNF-α were lower in the ADHD group than in the HC group. Our results indicate that the association between gut microbiome dysbiosis and immune dysregulation may contribute to the pathophysiology of ADHD.In summary, a total of three differentially abundant taxonomic clades at the genus level (Supplementary Fig. 1.Supplementary Fig. 2.Supplementary Fig. 3.Supplementary Fig. 4.Supplementary Fig. 5.


Objective: Cognitive and motor disintegration and other functional disturbances in various neuropsychiatric disorders may be related to inhibitory deficits that may manifest as a persistence or re-expression of primitive reflexes and few recent data suggest that these deficits may occur in Attention Deficit and Hyperactivity Disorder (ADHD).Methods: We have tested a hypothesis to which extent ADHD symptoms and balance deficits are related to persisting primitive reflexes, such as Asymmetric Tonic Neck Reflex (ATNR) and Symmetric Tonic Neck Reflex (STNR) in 80 medication-naïve children with ADHD (40 boys and 40 girls) in the school age (8–11 years) and compared these data with a control group of 60 children (30 boys and 30 girls).Results: These data show new finding that ADHD symptoms and balance deficits are strongly and specifically associated with persistent ATNR in girls and STNR in boys.Conclusions: These results provide first evidence in medical literature that ADHD in girls and boys is specifically related to distinguished neurological developmental mechanisms related to disinhibition of primitive reflexes. According to recent findings functional disturbances due to various insults such as brain damages, toxic influences or psychological stress may manifest in various stages of development and an early brain insult or trauma may influence pathological changes that may manifest later in life , 2. A spThese developmental disturbances may be understood within the concept of “dissolution” proposed by Hughlings Jackson, who proposed that later developed functions during ontogenesis of the central nervous system (CNS) tend to replace the older ones , 8. ThisTo the best of our knowledge there is no evidence in the scientific literature about relationships of ADHD symptoms, persistence (or re-expression) of the primitive reflexes and balance deficits. With respect to these findings we have tested a hypothesis to which extent ADHD symptoms are related to persisting primitive reflexes ATNR and STNR, and balance deficits in 80 medication-naive children with ADHD (40 girls and 40 boys) in the school age (8–11 years). For comparison of these data we also assessed a control group of 60 children (30 boys and 30 girls).In collaboration of the Center for Neuropsychiatric Research of Traumatic Stress and the ELSPAC study  data of 80 medication-naive children, including 40 girls  and 40 boys , with ADHD diagnosed according to DSM-IV criteria were collected. The collected data included scores of primitive reflexes ATNR and STNR, balance deficits measured as soft neurological signs and psychometric measure of ADHD symptoms. All the patients were medication-naïve and their assessments were done before starting treatment. The ADHD diagnosis was combined hyperactive-inattentive subtype with occurrence of ADHD based learning difficulties. Exclusion criteria were other child psychiatric and neurological disorders, obesity and metabolic disorders, substance abuse and also previous exposure to stimulant medication, left-handedness and intelligence lower than average according to Wechsler Intelligence Scale for Children (WISC-IV). Recruitment was based on consecutive visits of the untreated patients in the clinic  and the control group was recruited through school advertising in the same cities. The control group of 30 girls  and 30 boys  without ADHD fulfilled the same exclusion criteria. Parents of all the participants gave written informed consent and the research was approved by Charles University, First Faculty of Medicine Ethical committee in collaboration of the project ELSPAC and Charles University research project of the Center for Neuropsychiatric Research of Traumatic Stress.For assessment of balance deficits we have used four items from the Physical and Neurological Examination for Soft Signs scale (PANESS) by Denckla  that canStanding eyes closed- Item 9 “Sustentation Postures/Stations- “Now I want you to put one foot in front of the other, just as you did for walking the straight line forward and backwards right now, and close your eyes. Stay that way as long as you can, or until I tell you to relax.” Time with stopwatch duration of success up to 20 sec. Score: Use stopwatch-determine time to score in groupings (see form) of duration. Code for tendency to fall (1) and for bringing arms up and out to balance (1). Scoring is on the scale 0, 1, 2, 3.Walking Forward-Item 7 Tandem Walking: “Now be sure you put your heel against your toe and walk to the end staying on the line.” Score: An error consists of not placing the heel to toe or missing the line completely. Code number of gaps, misses, failure to place heel to toe. Scoring is on the scale 0, 1, 2, 3.-Item 8 Tandem Walking: “Now do the same thing backwards.” Score as in test for forward tandem gait on the scale 0, 1, 2, 3.Walking Backward-Item 7 Tandem Walking- the same procedure and scoring with eyes closed. Score on the scale 0, 1, 2, 3. The measurements of balancing deficits was done twice by two independent examinators and averaged.Walking ForwardAsymmetric tonic neck reflex (ATNR) usually measured by Schilder Test  presentsSymmetric tonic neck reflex measured by Bender-Purdue Reflex Test  merges 6The measurements of the primitive reflexes were done twice by two independent examinators and averaged.Frequently used measure of ADHD symptoms is Conners' Parent Questionnaire (CPQ) . The CPQThe questionnaire enables to calculate total score and it has also subscales based on factor structure of the questionnaire—I. Conduct problems ; II. Anxiety ; III. Impulsivity-hyperactivity ; IV. Learning problems ; V. Psychosomatic difficulties ; VI. Perfectionism ; VII. Antisocial behavior ; VIII. Muscular tension . The symptoms are rated on a 4-point Likert scale (from 0 to 3) by either one or both parents of the child. In this study CPQ was completed by child's mother and the responses were communicated with a psychologist performing the examination to assess their validity.Statistical evaluation of scores of the measures of ATNR, STNR, balance deficits and ADHD symptoms included descriptive statistics, Kruskal-Wallis ANOVA and Spearman correlation coefficients. To prevent Type II error which would disable to reject null hypothesis that ADHD symptoms are not linked to balancing deficits and primitive reflexes we performed Power Analysis and assessed the effect sizes characterizing correlation coefficients. The statistical evaluations were performed using the software package Statistical Version 6.CPQ) are significantly correlated with scores of persisting primitive reflexes ATNR in girls  and with scores of persisting primitive reflexes STNR in boys .The main results of this data analysis show that ADHD symptoms measured by total score of the Conners' Parent Symptom Questionnaire , walking forward  and walking forward with closed eyes just in boys . Other correlations of CPQ subscales with primitive reflexes and balance deficits are summarized in Other results indicate that CPQ total score is also significantly correlated with scores of balance deficits during Standing with eyes closed are significantly correlated with scores of persisting primitive reflexes ATNR in girls  and balance deficits during Walking fwd with eyes closed are significantly correlated with scores of persisting primitive reflexes STNR in boys . Other correlations of balance deficits with scores of primitive reflexes were not statistically significant  of the same age, who were assessed for CPQ, ATNR, and STNR scores and balance deficits. The control group of children was without ADHD symptoms. From the control group 2 boys manifested mild ATNR response and 1 girl and 2 boys manifested STNR response . Other children did not manifest ATNR or STNR response. The control group also manifested very rare and minor balance deficits that on the scale from 0 to 3 had maximum value 1, which occurred in 3 girls and 4 boys in standing with eyes closed, in 1 boy during walking forward, in 1 girl and 1 boy during walking back, and in 3 girls and 6 boys during walking back with eyes closed. Other children did not manifest balance deficits. The groups of girls and boys in statistical analysis using Kruskal-Wallis ANOVA were not statistically different with respect to age and intelligence.Most significant results of this study show that levels of ADHD symptoms and balance deficits in girls are associated with persisting ATNR, whether ADHD symptoms and balance deficits in boys are closely related to persisting STNR. These data indicate that ADHD in boys and girls are linked to different neurological developmental pathways that are related to specific disinhibition and persistence of these primitive reflexes, ATNR in girls and STNR in boys.In agreement with these results recent findings show that high proportion of children with ADHD manifest altered balance and motor abnormalities , 4. AccoThe novel findings of this study show that cognitive and motor dysfunctions, and disturbed balance are strongly associated with persisting primitive reflexes (ATNR in girls and STNR in boys) that were not sufficiently suppressed and inhibited in later stages of development. According to recent evidence persistence or re-expression of primitive reflexes may occur in various neuropsychiatric syndromes such as schizophrenia, various forms of dementia , 22, 23 In summary, results of this study show that ADHD symptoms and balance deficits are gender-specifically related to the persisting primitive reflexes ATNR and STNR. This relationship to the best of our knowledge represents the first documented finding that ADHD development in boys and girls is related to neurologically different developmental pathways that specifically influence disinhibition of ATNR girls and STNR in boys. Together these data indicate that ADHD symptoms may represent the process related to “neural dissolution” and “interference” of more primitive neural mechanisms, such as primitive reflexes ATNR and STNR, with higher levels of brain functions due to insufficiently developed cognitive and motor integration. In this context, gender differences in these manifestations might be related to specific and delayed brain maturation in boys compared to girls .These documented neurological differences related to gender-specific ADHD neurodevelopmental mechanisms need further research that may enable to develop new gender-specific neurorehabilitation methods for ADHD therapy focused on integration of primitive reflexes, as for example application of movement intervention programs , 24. TheAll datasets generated for this study are included in the article and/or the supplementary files.The study was approved by Charles University Ethical Board.PB, JK, and JR: collecting data, data analysis, and writing the manuscript. All authors contributed to the article and approved the submitted version.The study was supported by projects Progress provided by Charles University.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.


Attention deficit hyperactivity disorder (ADHD) is a condition that usually has its onset in childhood. Although the disorder persists into adulthood in half of cases, adult ADHD is often not recognized due to different psychopathological characteristics, quite often overlapping with other diagnoses such as mood, anxiety and personality disorders. This is especially true for bipolar disorder (BD), which shares several symptoms with adult ADHD. Moreover, besides an overlapping clinical presentation, BD is often co-occurring in adults with ADHD, with comorbidity figures as high as 20%. This review will focus on the comorbidity between ADHD and BD by exploring the magnitude of the phenomenon and evaluating the clinical and functional characteristics associated with ADHD–BD comorbidity in adults. Finally, the review will address the implications of pharmacologically treating the ADHD–BD comorbidity, providing suggestions in how to treat these complex patients and addressing the issue of treatment-induced manic switch with the use of stimulants and other medications for ADHD. Attention deficit/hyperactivity disorder (ADHD) is a typical childhood-onset disorder characterized by a deficit of attention and motor hyperactivity leading to significant impairment in academic/occupational, familiar and social functioning . In mostADHD has an estimated childhood prevalence of 4–7% , while iIn adults with ADHD the clinical expression of core symptoms is different compared with that observed in children and adolescents . Adults Another issue that further complicates the diagnostic process in ADHD adults is the high frequency of comorbid disorders, particularly mood, anxiety, personality and substance use disorders. Among these, bipolar disorders (BD) have overlapping symptomatology that may be frequently mistaken for ADHD, thus substantially contributing to its underdiagnosis and subsequent undertreatment.Therefore, due to the many similarities between adult ADHD and BD and the need for clinical guidance in the diagnosis and management of both conditions, we aimed at reviewing the published literature on adult ADHD comorbidity with an in-depth analysis of BD comorbidity. Lastly, we discuss the implications and issues that arise when treating adults with co-occurring ADHD and BD.Several studies have found a high prevalence of comorbid psychiatric disorders in adult ADHD ,10,11. TThe most frequently represented psychiatric comorbidities are mood and anxiety disorders, substance use disorders (SUD) and personality disorders, with rates for at least one comorbid psychiatric disorder ranging from 57 to 92% ,15,16. OMood disorders are especially common in patients with adult ADHD. A large epidemiological study conducted in 20 countries found a 12-month prevalence of major depression in the 15% of adult subjects diagnosed with ADHD . ClinicaAnxiety disorders are also very common in adults with ADHD, as documented in several studies that reported prevalence rates of anxiety disorders of around 50% ,16,20. ASeveral studies have investigated the association between ADHD and personality disorders, finding rates of comorbidity ranging from 10 to 75% in adult ADHD samples ,26,27,28Similarly, the diagnosis of ADHD put patients at risk for substance abuse and eventually the development of SUDs. Both subjects with ADHD and substance abuse are at increased risk for the other condition, although it is believed that ADHD more often predates the onset of SUDs, since illicit substances are often used as self-medication in the effort to deal with ADHD symptomatology . The mosBipolar disorder (BD) is characterized by the alternation of mood episodes and variable periods of euthymia, which lead to worse overall functioning and quality of life. Several symptoms encountered in ADHD are common in BD, particularly during elated phases, making differential diagnosis often challenging. However, the existing differences in the phenomenology of the two disorders help guide the clinicians out of the maze. The core ADHD dimension of inattention is present during mood episodes; however, in ADHD it is frequently reported as a tendency to wander from one thought to another, whereas during hypomanic episodes it is often described as a peculiar clarity of thoughts, and during manic episodes it is the consequence of thought acceleration, leading to the typically observed distractibility. The impulsive dimension is also commonly found in BD during euphoric phases, although in those cases it can be often drawn back to inflated self-esteem or grandiosity, eventually leading to underestimating the consequence of actions. Similarly, the inability to stop and relax typical of ADHD can be observed in patients with BD during both anxious depression and manic episodes, although in the latter it usually takes the shape of increased goal-directed activity.Some other clinical characteristics can help clinicians in differentiating BD from ADHD. First, some intraepisodic features typical of BD are not observed in ADHD: for instance, the increase in sexual activity often reported during manic phases is not reported in ADHD. Furthermore, sleep problems express differently: sleep deprivation in ADHD, whenever it occurs, is usually accompanied by discomfort and fatigue; on the other hand, during manic phases sleep need is reduced, oftentimes without any concomitant physical discomfort. Finally, psychotic symptoms, which can occur during severe mood episodes, are not observed in patients with ADHD, regardless of the severity of the disorder .Course characteristics also help disentangling BD from ADHD. First and most importantly, BD has an oscillating course, with phases clearly different from one another. Therefore, in patients with BD, symptoms such as distractibility, impulsivity and hyperactivity fluctuate over time. On the contrary, ADHD symptomatology is usually stable over time . SecondlHowever, besides diagnostic overlapping, ADHD and BD often co-occur, as demonstrated by several studies. The National Comorbidity Survey-Replication (NCS-R), one of the broadest and most representative mental health survey conducted in the USA, estimated the prevalence of ADHD in 4.4% of 3199 respondents. Among those, comorbid BD could be diagnosed in the 21.2% . In EuroClinical studies also reported high comorbid figures between ADHD and BD. In an early report on the first 1000 participants of the Systematic Treatment Enhancement Program for BD, the researchers found that comorbid ADHD could be diagnosed in 9.5% of BD patients, a figure that rose to 14.7% in males. Comorbid anxiety and SUDs, together with a greater burden of disease, were more frequent in patients with both conditions . McIntyrSeveral longitudinal studies have tried to assess whether children with ADHD would develop (hypo)manic episodes over time. In the first, a 10-year follow-up study, boys with ADHD had a 7.9-times higher morbidity risk than healthy controls for developing BD by the age of 21 . In a siSome studies assessed ADHD–BD comorbidity according to the type of BD. The above-mentioned population studies, the NCS-R and WHO collaborative study, reported very similar ADHD comorbidity rates among patients with BD type I and II ,49. ClinIn conclusion, the reviewed clinical studies found that around 10–20% of adult patients with BD has comorbid ADHD, with even higher rates in those with earlier age at onset . The aboBipolar disorder is a psychiatric condition particularly at risk for suicidal behaviors and committed suicide. On the other hand, ADHD can also increase suicide risk ,61,62. HSince not stabilized BD can express with mood swings, impulsivity, hyperactivity and inattention, symptoms that overlap with ADHD presentation, the first goal of ADHD–BD comorbidity treatment should always be mood stabilization. Moreover, after being properly stabilized, some ADHD/BD patients might even not fulfill diagnostic criteria for ADHD anymore . HoweverEarly reports suggest a higher risk of treatment-emergent manic episodes in patients with ADHD treated with MPH, although more recent studies mitigated this association. In a retrospective study, DelBello and colleagues found an association between a past history of treatment with stimulants and an earlier onset of BD in a group of adolescents with ADHD, suggesting that stimulant exposure could accelerate the onset of BD . In anotRegarding atomoxetine, in a first retrospective chart review on 7 pediatric patients with BD and ADHD already treated with mood stabilizers, no hypomania or mania was reported with the use of adjunctive atomoxetine over mood stabilizers for a period up to 18 months . SimilarBesides MPH and atomoxetine, amphetamine salts were studied in patients with ADHD–BD comorbidity. In a first study, youth patients with manic symptoms were initially treated with divalproex sodium for 8 weeks, showing a reduction of manic symptoms but no effect on ADHD symptomatology. At the end of this open-label phase, youths randomized to mixed amphetamine salts in adjunct to divalproex showed a significant reduction of ADHD symptoms, with only one patient experiencing a worsening in manic symptoms . More reAdult ADHD is often characterized by symptoms such as impulsivity, distractibility and restlessness, which clearly overlap with BD symptomatology, thus making differential diagnosis between the two disorders a challenge. Moreover, ADHD and BD often coexist, as highlighted by several population and clinical studies. Adults with ADHD and comorbid BD are a particularly critical group of patients showing a severe and burdensome clinical picture, with a lower quality of life, a higher number of mood episodes, an increased prevalence of substance abuse and dependence, and a worse overall functioning. In spite of that, only a few studies have investigated the best treatment modalities in these complex patients, therefore recommendations should still be considered as preliminary. In short words, when ADHD and BD co-occur, mood stabilization should be the first goal of treatment. When a mood stabilizer is in place, the augmentation with stimulant medications is effective in ameliorating the ADHD symptomatology. In these cases, the possibility of a treatment-emergent (hypo)manic switch should be taken into account, although emergence is uncommon when proper mood stabilization is in place. Finally, adults with ADHD and comorbid bipolar depression may benefit from the use of lisdexamfetamine.


Attention-deficit/hyperactivity disorder (ADHD) is associated with a broad range of physical health problems. Using different research designs to test whether ADHD has a causal role in these associations is important because comorbid health problems increase the serious social and economic impacts of ADHD. We used 2-sample Mendelian randomization (MR) to infer causal relationships between ADHD and previously implicated physical health conditions. Different MR methods were used to test the robustness and plausibility of our findings. Consistent findings underwent bidirectional and multivariable MR. We found evidence of ADHD having a causal effect on childhood obesity  and coronary artery disease  with consistent results across MR approaches. There was additional MR evidence for a bidirectional relationship between ADHD and childhood obesity. The relationship with coronary artery disease attenuated when controlling for childhood obesity. There was little evidence for inferring a causal effect on other cardiometabolic, autoimmune, allergic, and neurological diseases. Our findings strengthen the argument for effective treatment of children with ADHD, and suggest that clinicians who manage ADHD need to be aware of the risk of childhood obesity to reduce future risks of coronary artery disease. ADHDattention deficit hyperactivity disorderBMIbody mass indexCADcoronary artery diseaseCIconfidence intervalGWASgenome-wide association studyIVWinverse variance weightedMRMendelian randomizationMVMRmultivariable Mendelian randomizationSNPsingle-nucleotide polymorphismAttention-deficit/hyperactivity disorder (ADHD) typically begins in early childhood and has a worldwide prevalence of approximately 5% in school-aged children , 2. ApprThere is growing evidence from case-control and cohort studies that ADHD is associated with a broad range of physical health problems. These include obesity , type 2 Alternative research designs are needed to infer causation; Mendelian randomization (MR) offers an approach by reducing bias from confounding and reverse causation . The ratP < 1 ×10−7 (Web Table 1) (available at https://doi.org/10.1093/aje/kwaa273). For physical health outcomes, SNPs were identified from GWAS summary statistics of European ancestry populations for cardiometabolic factors   as proxies for both ADHD (exposure) and physical health (outcomes). For ADHD, 13 SNPs were identified from a GWAS of individuals of European ancestry   at P < 1ex (BMI) ; childhoex (BMI) ; coronarex (BMI) ; myocardex (BMI) ; hyperteex (BMI) ; systoliex (BMI) ), neurolex (BMI) , epilepsex (BMI) ), autoimex (BMI) ), allergex (BMI) , asthma ex (BMI) , eczema ex (BMI) ), and luex (BMI)  (detailsex (BMI) . GWASs fex (BMI) . SNPs asTwo-sample MR was conducted to investigate the causal role of ADHD on physical health outcomes, using GWAS summary statistics to assess both SNP-exposure  and SNP-outcome  associations. This allows the estimation of an unconfounded causal effect between exposure and outcome, if certain assumptions  hold truThe SNP-exposure and SNP-outcome associations were assessed using 3 methods with different assumptions: inverse-variance weighted approach (IVW) , weighteOdds ratios for associations between binary exposures and binary outcomes in 2-sample MR studies are interpreted as the odds ratio for outcome per unit increase in the log odds ratio of the exposure. When examining binary exposures in MR settings, causal inferences are valid for the continuous liability underlying the binary exposure . Thus, wP values that have not been corrected for multiple testing (while acknowledging the number of correlated phenotypes that have been tested) and focus on consistent results across MR methods to assess the strength of evidence favoring a causal effect.We present MR F statistic (F > 10 suggests results should not suffer from weak instrument bias)  (Instrument strength (first MR assumption) was assessed using the nt bias) . Heteroggeneity) ; where hgeneity) .P < 1 ×10−6  and ADH 5 ×10−8 .Multivariable MR (MVMR) analyses were conducted to assess 2 potential mediators of the association between ADHD and CAD: childhood obesity  and lifeAll steps of the 2-sample MR we performed are summarized in MR results using the IVW approach are shown in There was little evidence of a causal effect of ADHD on BMI, myocardial infarction, hypertension, systolic blood pressure, type 2 diabetes mellitus, migraine, epilepsy, autoimmune and allergic diseases, and lung cancer. There was evidence of a causal effect from MR-Egger regression  for inflammatory bowel disease (Web Table 4).F statistics calculated for all the genetic variants used as instruments (Web Table 5). Heterogeneity in IVW was detected for BMI, hypertension, systolic blood pressure, and allergic rhinitis (Web Table 5). None of the leave-one-out plots identified any outlying SNPs . There was no evidence that the MR-Egger intercept differed from the null for any of the heterogeneous tests, suggesting that the detected heterogeneity was unlikely to be due to bias from directional horizontal pleiotropy (Web Table 6).Good instrument strength was indicated by the Based on these initial findings, there was some evidence of a causal effect of ADHD on childhood obesity, CAD, and inflammatory bowel disease. Hence, these 3 outcomes underwent additional sensitivity analyses (Web Appendix 2). Evidence of a causal effect was detected through simulation extrapolation–adjusted MR-Egger regression for childhood obesity and CAD; hence, we also investigated possible bidirectional effects. We did not detect evidence of a causal effect for inflammatory bowel disease through simulation extrapolation adjusted MR-Egger regression , which suggested pleiotropic effects; hence, it was not taken forward for further analyses.We performed bidirectional MR to examine the effect of CAD and childhood obesity on ADHD . There wP < 5 ×10−6)  were in linkage disequilibrium with any of the 8 SNPs associated with childhood obesity (5 ×10−6) , suggestF statistic was >10, indicating good instrument strength (Web Table 9).Because obesity and smoking are established risk factors for CAD  that areF statistic was <10 (Web Table 10), which indicates low instrument strength and which suggested genetic overlap between ADHD and lifetime smoking heaviness; hence, we could not disentangle the effects of ADHD and smoking heaviness on CAD.The (univariable) causal effect of ADHD on lifetime smoking heaviness was 1.07 . When genetic variants for ADHD and lifetime smoking heaviness were simultaneously entered in the MVMR model, the direct causal effect of ADHD on CAD remained stable , whereas the effect of lifetime smoking heaviness on CAD was attenuated  (Web Table 10). As shown in In this study, we used a 2-sample MR approach to test putative causal effects of ADHD on physical health outcomes, using genetic variants as instrumental variables as proxies for ADHD. We found evidence consistent with a causal effect of ADHD on CAD and evidence for a bidirectional association between ADHD and childhood obesity. There was little evidence of ADHD causal effects on neurological, autoimmune and allergic diseases, or lung cancer. MVMR results suggested that the causal effect of ADHD on CAD is  mediated by childhood obesity.As reported previously, in observational studies , 20, 43,Many studies have shown associations between ADHD and obesity in adolescents and adulthood , 46, 47.Bidirectional MR for ADHD and childhood obesity suggested possible effects in both directions, in line with a previous MR study. Maternal effects have been discussed in depth by Martins-Silva et al. , who fouF statistic was <10, indicating genetic overlap between ADHD and lifetime smoking heaviness. This is not altogether surprising, because smoking initiation (a core component of the lifetime smoking instrument) is highly correlated with impulsivity and risk taking  from the ADHD GWAS to increase the number of instruments so that sensitivity analyses, such as MR-Egger regression, could be performed (3 included SNPs were not genome-wide significant). Because relaxing the threshold for SNP inclusion increases the risk of potential sources of bias, such as pleiotropy or heterogeneity, we deemed increasing the P value threshold further would not to be beneficial. Results of our sensitivity analyses suggested there was substantial heterogeneity in MR-Egger regression, which was a marker of measurement error in the instruments. This indicated that there might not have been enough power to detect causal associations, because weak instruments bias associations toward the null in 2-sample MR studies (We relaxed the  studies . In addi studies .P < 0.0025 (0.05/15), which is likely to be too conservative in this context, and could still possibly be chance findings. Furthermore, although there was little statistical evidence for horizontal pleiotropy, the underlying biological pathways leading to ADHD are unknown for most of the genetic variants and, therefore, the possibility of pleiotropic effects of these variants cannot be discounted. Because in these analyses we used a 2-sample MR framework, which is based on publicly available data, we were not able to test whether the genetic variants used as instruments are independent of potential confounders of the observed exposure to outcome associations. Confounding may also arise due to population stratification   , assortalations) , or selelations) .In conclusion, using 2-sample MR, we found evidence in favor of a causal effect of ADHD on CAD that is potentially mediated by childhood obesity. Additional research focusing on long-term follow-up of the physical health of children with either a high number of ADHD symptoms or an ADHD diagnosis is required to elucidate these relationships. Our findings strengthen the argument for early and effective treatment of ADHD symptoms in children because the symptoms may also have an impact on later physical health.Web_Material_kwaa273Click here for additional data file.


Likewise, a significant moderate positive correlation  was found between the HF n.u. and sensory profile total scores among children with ADHD. Children with ADHD have significantly lower resting state parasympathetic functions compared to their typically developing peers.Previous studies suggest that parasympathetic functions support sensory behaviours. However, the relationship between sensory behaviours and parasympathetic functions remain inconclusive and inconsistent among children with and without attention-deficit hyperactivity disorder (ADHD). This research aims to examine the sensory behaviours and resting parasympathetic functions among children with and without ADHD. We compared sensory behaviours and baseline parasympathetic functions of 64 participants, with 42 typically developing and 24 ADHD male children aged 7–12 years. Sensory behaviours were evaluated using the sensory profile. Baseline parasympathetic functions were indexed using the normalized unit of heart rate variability high-frequency bands (HF n.u.). Children underwent an experimental protocol consisting of watching a silent cartoon movie while HF n.u. is continuously monitored, within a controlled environment. The results of this research showed significantly lower HF n.u. ( Attention-deficit hyperactivity disorder (ADHD) is a childhood neurobehavioural disorder characterized by persistent inattention, hyperactivity, and impulsivity. The prevalence of ADHD has been suggested to reach between 5.29% and 7.1% in children and adolescents and at 3.4% (range 1.2–7.3%) in adults . NeverthThe ANS, specifically its parasympathetic (PNS) branch, has been linked with ADHD behavioural manifestations –12. SevePrevious studies suggested that the PNS functions are related to how children with and without ADHD regulate responses to sensory stimuli , 16. ComChildren with ADHD have been found to have atypical behavioural and physiological responses to sensory stimuli , 22. OneIn this study, we follow salient recommendations suggested by Gomez et al.  in usingThis research aims to examine the sensory behaviours and resting parasympathetic functions among children with and without ADHD. Specifically, this research sought to: (1) determine differences in sensory behaviours between typically developing children and children with ADHD; (2) determine differences in resting parasympathetic functions between typically developing children and children with ADHD; and (3) determine the relationship between sensory behaviours and resting parasympathetic functions among typically developing children and children with ADHD.A descriptive cross-sectional study will be used in this research. Ethical approval was obtained from the University of Santo Tomas-Graduate School, Ethics Review Board. Before data gathering procedures, assent forms and parental consent forms were distributed to the participants and their respective parents. Whenever discomfort could not be tolerated by the child, the testing procedure was stopped.M = 8.90 yrs + SD = 1.52) and 24 ADHD (M = 9.40 yrs + SD = 1.71) children. Schaaf et al. (2003) previously suggested that at least n = 20 for each group  is an adequate number for comparison of differences in PNS functions. For both groups, the mother was the primary caregiver with at least a college-level educational background.A convenient sampling technique was used to recruit participants in this research. To recruit children with ADHD, invitation letters were sent to therapy centres. Typically developing (TD) children were recruited through partner primary schools of the university. Participants must be males, aged 7–12 years, currently attending a primary school, and have no known history of cardiorespiratory, orthopaedic, or metabolic conditions. TD children have no known history of behavioural, psychological, or developmental disability, while children with ADHD had no comorbid behavioural, psychological, or developmental disability apart from ADHD. A total of 64 participants were recruited in this study, with 42 typically developing  and the Participation and Environment Measure for Children and Youth (PEM-CY). The SP is a behavioural checklist that measures how children respond to sensory stimuli as they participate in daily activities . The PEMHeart rate variability (HRV) enables this research to look at the physiological activity of the PNS and their interactions across different experimental conditions . Specifically, the HF band of HRV will be used to represent PNS functions. For this study, a Polar H2 Heart Rate Monitor  was used to measure heart rate. The equipment consists of a heart rate monitor that is attached to a chest belt and transmits heart rate signals via an infrared device to a computer notebook. Heart rate monitors such as the Polar H2 allows efficient data acquisition of heart rate variability and is suitable for the paediatric population. There is some evidence that supports the use of Polar heart rate monitors as a valid instrument to measure heart rate variability .The experimental paradigm used in this study was adapted from the resting baseline condition of previous similar studies, summarised by Gomez et al. . The expDuring the day of experimental procedures, participants were greeted by the researcher and led inside the testing room, initially accompanied by the parent where general instructions were given. With the help of the parent, the Polar H2 chest strap was placed just below the chest of the participant. The researcher instructed the child to sit on a comfortable child-sized chair, facing a 19-inch monitor three feet away. A set of over-the-ear closed-back headphones was used to control extraneous noise. During the actual experimental procedures, the room was dimmed to 10 lux (illumination level), temperature set to 23–25°C, humidity level at 60–80%, and background noise level at 40–45 decibels. The researcher was located 60–80 cm away to the left of the participant, keeping interaction minimal. Testing procedures in this research were adapted from a similar previous study . EnvironThe aHRV  software was used to analyse HRV data employing the current standards and guidelines for HRV reporting . The spet-test. Furthermore, a Pearson product-moment correlation coefficient was computed to assess the relationship between behavioural and physiological measures related to the regulation of response to sensory stimuli. SPSS 23.0 was used for all statistical analyses, and the significance level is set at a critical level of 0.05.Descriptive statistics involving measures of central tendencies and variation are used to describe the salient characteristics of the data gathered. Computed composite scores on the behavioural questionnaires and the HF n.u. were tested for significant differences between children with and without ADHD using an independent The results of this study are divided into three sections and are summarised in n = 64). Typically developing children showed significantly higher (t(64) = 14.13 = p < 0.01) SP scores (M = 523 + SD = 49.16) compared to children with ADHD (M = 356 + SD = 40.49). No significant differences were found for PEM-CY scores. Both participants engage in a similar frequency of participation in daily activities; however, children with ADHD exhibit significant sensory behaviour problems when facing such activities.We compared group differences on the composite scores on sensory processing and participation frequency among children with and without ADHD  and ADHD  children. This research finds significantly lower baseline parasympathetic functions for children with ADHD compared to their typically developing counterparts (t(64) = 7.84, p < 0.01).Resting-state parasympathetic functions, as represented by the HF n.u., were compared between typically developing  was found between the HF n.u. and SP total scores among children with ADHD.Behavioural and physiological measures were correlated independently for each group. A significant moderate positive correlation (Taken together, the results of this research show that children with and without ADHD participate in daily activities with similar frequency, but those with ADHD experience more problems approaching sensory stimuli with lower parasympathetic functions. More specifically, ADHD children with higher parasympathetic functions exhibit better sensory processing.The purpose of this study was to examine behavioural and resting parasympathetic functions among typically developing children and children with ADHD. Previous studies found sensory processing difficulties among children with ADHD , which cADHD presents with several behavioural issues, which have been previously linked with an underlying neurological basis. We know from previous research that children with ADHD may have compromised sympathetic functions related to behavioural response to sensory stimuli , 21. TheThe results from this study provide a novel inquiry on how lowered PNS functions may affect how children with ADHD attain and sustain a calm and focused state that supports behavioural responses to sensory stimuli in daily activities. Our results espouse this idea, and we suggest that PNS functions may be a vital component in the evaluation of atypical sensory behaviours as well as other multifaced behaviours associated with ADHD.This research is not without any limitations. First, the ADHD sample in this study represents a general clinical population, which may vary in their sensory processing difficulties. Future studies may consider controlling the specific subtype of ADHD and describing their certain sensory issues. Second, while the resting-state parasympathetic functions describe a child's ability to respond, it will be interesting to examine the responsiveness to sensory stimuli by considering a sensory stimulation block to the experimental paradigm. Lastly, the use of a single autonomic measure may not depict a composite physiological response; hence, we recommend describing coexisting sympathetic functions.In summary, this research provides evidence supporting significantly lower resting-state parasympathetic functions in children with ADHD compared to their typically developing peers. The results in this research contribute to the growing body of literature supporting the neurophysiological evidence related to the pathology of ADHD. The link between behavioural and physiological measures suggest neurophysiology-based outcome measures and interventions can be considered in addressing symptoms of ADHD. Future studies should address better sampling design, controlling for specific ADHD subtypes, investigate the ability to respond to sensory stimuli, and describe coexisting sympathetic functions.


Modern plant-breeding practices have narrowed the genetic base of wheat, such that there is a need to introduce new germplasms with underexploited diversity into breeding programs. Wheat landraces are a very valuable resource when searching for genetic variation, which not only possess increased adaptability, but also quality-related traits. Several studies have shown a wide genetic diversity in Spanish wheat landraces compared to other germplasm collections; therefore, the main objective of this study is to analyze the variability in a collection of 189 landraces from the Spanish National Plant Genetic Resources Centre , in relation to end-use quality traits. We characterized the whole collection for high-molecular-weight glutenin and puroindoline allelic composition, and for gluten strength. In addition, grain protein content, grains per spike, and thousand kernel weight were evaluated in samples from four-year field trials. The relationship between glutenin composition and quality was evaluated, and some alleles strongly associated with high quality were identified in the collection, some of them specific for Iberian landraces. The results also show the presence of novel variability within high-molecular-weight glutenin and puroindolines, which needs to be characterized further in order to assess its influence on wheat quality. In addition, a set of landraces showing outstanding values for gluten quality and a good agronomic performance was selected for testing in field trials in order to evaluate the suitability of their direct use in cropping systems. Triticum aestivum spp. vulgare (Vill.) , which represents roughly 90% to 95% of the total production. Bread wheat is an essential crop for humans; it provides 20% of total protein and calories in daily intake through a variety of geographically derived products  [Wheat is one of the three main crops grown across the world. It covers 214 million hectares and represents a quarter of total cereal production, with 765 million tons produced in 2019 [s, etc.) . Currents, etc.) ,9,10,11.Glu-1 loci, located close to the centromere on the long arm of homoeologous group 1 chromosomes  [Glu-A1 locus encodes one or no subunit, the Glu-B1 locus encodes one or two subunits, and the Glu-D1 locus encodes two subunits [Glu-D1d, Glu-B1b, and Glu-B1c alleles, strongly associated with high end-use quality, have been described as the most suitable to breeds for end-use value [Although the main current breeding goal is the development of stable yield cultivars, most wheat production is used for human consumption and, thus, wheat end-use quality is always a key breeding target . The mai Glu-D1) . These l Glu-D1) ,18. In gsubunits ; therefosubunits ,20,21, asubunits ,22. The se value . Howeverhardness locus (Ha), located on the short arm of chromosome 5D: the Pina-D1 gene and the Pinb-D1 gene, which code for the puroindoline a and puroindoline b proteins, respectively [Besides endosperm proteins, endosperm texture (or grain hardness) is also an important quality factor in bread wheat. Kernel texture, directly related to the amount of damaged starch produced during the milling process, is used to classify wheats as either hard or soft. Hard wheat grains produce more damaged starch, which results in higher water absorption when doughs are formed ,25 , conserved in the Spanish National Plant Genetic Resources Centre, Centro de Recursos Fitogenéticos, INIA, Alcalá de Henares (CRF-INIA). Given the high agroclimatic diversity of the Iberian Peninsula, these landraces, which are locally adapted, represent a large source of genetic variability [In Spain, the widest collection of wheat landraces is composed of 522 Spanish accessions of iability ,40,41. Miability ,43. A reiability . Thus, tThe germplasm bank codes of the studied landraces along with the commercial varieties’ names are given in In order to facilitate the exploitation of Spanish wheat landraces in breeding programs, we determined their allelic profile for quality-related genes: puroindolines and HMW-Gs subunits.a allele of the Pina-D1 gene, with the Pina-D1b hard-type allele only being present in eight accessions  allele Pinb-D1a (80.65%). The frequencies of the Pinb-D1b and Pinb-D1d hard-type alleles were low (2.69%). Two additional rare Pinb-D1 alleles were found. Fifteen landraces carried an allele previously described only in Spanish landraces and spelt wheat, not included in the Catalogue of Gene Symbols, but tentatively classified as the allele Pinb-D1ad [Pinb-D1 allele that was not previously described. This allele has a G/A change in position 55, which produces an A19T change in the predicted protein sequence. The puroindoline b protein sequence alignment can be found in Pina-D1a/Pinb-D1a combination, with the Pina-D1b, Pinb-D1b, and Pinb-D1d alleles being present in the same proportions.The allelic variation in puroindoline genes was determined in the full set by comparing the gene sequences to alleles available in the GenBank database. Most landraces (95.70%) carried the wild-type soft cessions . For theinb-D1ad . This alinb-D1ad . Three lGlu-1 homoeoloci  and PCR for the complex loci see . The lanGlu-A1 locus, fourteen different alleles at the Glu-B1 locus, and nine different alleles at the Glu-D1 locus were identified in the 189 Spanish landraces analyzed  found in 53.44% of the landraces, followed by subunits 2¨, 1, and null  that were found in 17.99%, 16.40%, and 11.64% of the landraces, respectively. The Glu-B1 locus was the most polymorphic, with nine previously catalogued alleles identified, namely a, d, e, f, aq, al, am, i, and u. Among them, e, f, and u were the most frequent . Other subunits/alleles were found at a low frequency . At the Glu-D1 locus, seven previously described alleles were found, with 2+12  being the most frequent combination, followed by 4+12 . The d, h, j, l, and u alleles were rare, being found in less than eight varieties.At the b allele at the Glu-A1 locus; the b, c, or d alleles at the Glu-B1 locus; and the d allele at the Glu-D1 locus  and gluten strength, measured by the sodium dodecyl sulfate-sedimentation (SDSS) test. These traits were evaluated in samples from four different seasons in landraces and three seasons in the reference set . ClimatiIn landraces, mean GPC was between 11.51% and 17.59%, being significantly higher than in the reference set varieties for the three years when both sets were sown . The higSpearman’s correlation analysis was carried out between traits and between years see . As expeThe effect of year and genetic structure  were found. Regarding this locus, the 7*+9 combination and i allele were the most outstanding allelic variants for gluten strength. A significant influence of the Glu-D1 genotype was also found, with significant differences among the a, c, d, and j alleles and with the d allele being associated with the highest gluten strength .The effect of the year and genetic structure of the collection on the TKW and GN values in the set of landraces was evaluated by an ANOVA test . No yearThe variability in climatic conditions between years significantly affected TKW, with values being higher in the rainiest season (2017–2018) and being much lower in the driest seasons (2016–2017 and 2018–2019). The genetic structure also had a significant effect on this trait. All populations performed significantly differently across years, and landraces belonging to genetic population 3 were those that achieved higher TKW values in every year. For GN, year also had a significant effect, corresponding to the highest values in the mild 2019–2020 season. Genetic populations 2, 3, and 4 showed similar values, with population 1 being the one with the significantly lowest values for any year.Glu-1 loci), two main quality determinants in bread wheat.In the present work, genetic variability related to end-use quality was studied in a set of 189 Spanish landraces by characterizing the genes controlling endosperm texture (puroindoline loci) and HMW-Gs composition  were found. Among all the Glu-B1 variants examined, the 7*+9 combination and i allele were the most outstanding alleles for gluten strength. These results confirm those obtained by other authors for the 7+9 subunits [f allele are of particular interest because this allele, as previously mentioned, is characteristic of Iberian landraces [Glu-D1 genotype was found, showing significant differences in the a, c, d, and j alleles. Glu-D1d was associated with the highest gluten strength, and c and l were associated with the lowest. The positive influence of the Glu-D1d allele compared to a and c has also previously been shown [c allele to be the one with the lowest influence on the sedimentation volume, but, here, the l allele showed even lower SDSS volume values. It should be highlighted that the influence of this allele has not been previously established, as it is a very rare allele. Further studies are required to determine the influence on gluten strength of the new alleles identified in the collection that could not be addressed due to the low number of landraces (only one or two) carrying them , which is in accordance with some authors ,64,74, w, which iAlthough breeding for quality must be a key target of a wheat breeding program, yield is a major determinant of crop value and the main goal of any program. The inclusion of landraces in quality breeding programs will be favored if they have good agronomic values. Field characterization can aid breeders when selecting local donor materials that ensure good end-use properties in the derived elite cultivars without high detriment to agronomic performance. In addition, although landraces perform worse than modern varieties in terms of yield, their yield stability can be higher since they are more resilient to adverse conditions and can have a better performance in unfavorable conditions ,87,88. IWith this aim, the collection of landraces besides the reference set was evaluated for agronomic performance in four-year trials, in which the yield components TKW and GN were evaluated. TKW is important because it not only determines the flour yield, but also affects the milling quality of wheat grains  and becaFor TKW and GN, as well as for GPC, the influence of the genetic structure was observed. This supports the existence of different gene pools in the landrace collection that can be explored further in relation to wheat improvement. It is interesting that landraces from population 3 showed high TKW and GPC values, in spite of the negative correlation found between these variables. This group of landraces have a diverse eco-geographical origin, but, with population 4, are the most genetically different from modern cultivars . Combining all the traits analyzed, a set of fifteen landraces with high SDSS values and high TKW and GPC values were selected to be characterized further and more deeply in field trials for agronomic performance and for other parameters related to end-use quality . This wiTriticum aestivum subsp. vulgare (Vill.) maintained in Spanish National Plant Genetic Resources Centre . All these accessions were homozygous lines derived from GenBank-original accessions, chosen based on their collection site data and agro-morphological characteristics to represent all of the different ecological and geographical areas of Spain [For this study, a set of 189 bread wheat landraces and old cultivars were selected from the Spanish national collection of of Spain . This seof Spain .http://webx.inia.es/web_inventario_nacional/Introduccioneng.asp, accessed on 23 March 2021).Additionally, this study included a set of 18 modern bread wheat varieties composed of the cultivars most widely grown in Spain during the last 50 years. A complete list of the materials is presented in For each accession, genomic DNA were isolated from young leaves of one plant using a modified CTAB method .Pina-D1 and Pinb-D1 genes were amplified as described in [Pina-D1a: NCBI ID KT885195; Pinb-D1a: NCBI ID KT885196; Pinb-D1b: NCBI ID KT885197; Pinb-D1d: NCBI ID KT885198; and Pinb-D1ad: NCBI ID JX187515) and translated to proteins for analysis with the same software.In order to characterize puroindoline genotypes, the ribed in . The PCRribed in . The genGlu-1 homoeoloci [The total set was also genotyped for the complex moeoloci , which emoeoloci  and fracmoeoloci . Some sumoeoloci ,72,102. moeoloci . All landraces were sown for four consecutive years. In the first year (season 2016–2017), the accessions were sown in an augmented design in plots of four rows (1 m long) in Alcala de Henares . In the following years , landraces and the reference set were sowed in the same conditions in the experimental fields of the School of Agricultural, Food, and Biosystems Engineering . Daily meteorological data were recorded over the period of study (autumn 2016 to summer 2020) at weather stations located near the growing areas. The average monthly precipitation and the minimum and maximum temperatures from October to June are shown in The number of Grains per Spike (GN) was recorded according to the International Board of Plant Genetic Resources (IBPGR) from at least five different spikes in each accession. After harvest, Thousand Kernel Weight (TKW) was estimated for all accessions, and a sample of 15 g was ground in a Tekator mill for quality analysis. Protein content on dry matter (GPC) was estimated by near-infrared reflectance analysis (NIR) using a PerCon Inframatic 8600 . Gluten strength was determined on 1 g of whole grain flour samples by an sodium dodecyl sulfate -sedimentation (SDSS) volume test, according to Dick and Quick (1983)  with minGlu-1 loci, the nonparametric Kruskal–Wallis  and Wilcox tests  were used. Alleles found in less than three landraces were excluded from this analysis.All statistical analyses were performed with the software R version 3.5.2 . Mean, sGlu-A1y and Glu-B1f, which is related to Iberian material, may be of particular interest in breeding for quality. The four-year trials allowed us to identify landraces with outstanding values. As this collection has been previously throughput genotyped [In this work, the genetic variability of quality-related genes was evaluated in a set of Spanish wheat landraces. New alleles for puroindolines and HMW-Gs subunits were identified, although the potential of this new variability needs to be studied further by a segregating populations analysis. Moreover, the influence of HMW-Gs variability in gluten strength revealed that some alleles that are not present in modern cultivars, such as enotyped , these d


Intracerebral hemorrhage (ICH) has limited therapeutic options. We have shown that an intravenous injection of human umbilical cord-derived mesenchymal stromal cells (hUC-MSC) 24 h after an ICH in rats reduced the residual hematoma volume after a moderate hemorrhage but was inefficient in severe ICH. Here, we investigated whether a treatment in the hyperacute phase would be more effective in severe ICH.Wistar rats were randomly selected to receive an intravenous injection of hUC-MSC or the vehicle 1 h after a severe ICH.The hyperacute treatment with hUC-MSC did not affect the 22-day survival rate, the motor function or the residual hematoma volume.These results indicate the need for optimization of hUC-MSC-based therapies for severe ICH. Hemorrhagic stroke, caused by the leakage of blood from blood vessels to the brain, is a life-threatening condition that reduces the quality of life of a large number of patients worldwide without effective treatments. Here, we induced a severe hemorrhagic stroke in rats to study the effects of a treatment using mesenchymal stromal cells, stem cells obtained from the umbilical cord tissue capable of producing protective molecules for the brain. The treatment; however, did not improve some aspects of the disease, such as the motor ability and the size of the brain lesion, indicating that further studies are still necessary. Intracerebral hemorrhage (ICH) is a common cause of neurological morbidity in adults, corresponding to around 26% of the cases of stroke . DespiteMesenchymal stromal cells (MSC) are multipotent cells that can be obtained from a variety of tissues and which can be induced to differentiate into cells of the mesodermal lineage . MSC constitutively secrete several trophic factors and inflammatory mediators, as well extracellular vesicles carrying biologically active molecules, which have been shown to have neuroprotective, antioxidant and immunomodulatory properties . MoreoveA total of 34 male Wistar rats aged 7–10 weeks (mean age: 57 ± 4 days) and weighing 244–305 g were used in this study. All procedures were approved and conducted in accordance with the Animal Care and Use Committee at the Universidade Federal do Rio de Janeiro (protocol number 111/14), and in compliance with the ARRIVE guidelines. All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the US National Academy of Sciences Guide for the Care and Use of Laboratory Animals.To model a severe ICH, 34 rats received an intrastriatal injection of 0.25 U collagenase IV-S  in the left striatum, as previously reported . Animals2. After reaching confluence, cells were enzymatically dissociated by incubation with a trypsin solution  for 5 min  = 0.2959, p = 0.5865).One animal from the ICH + hUC-MSC group died immediately after the hUC-MSC injection, a potential, but uncommon, complication of the iv. injection of MSC in rodents that had been reported before ,16. The 6 hUC-MSC was not able to improve motor function.The rotarod test was employed to assess motor function. Animals were pretested by an examiner blinded to treatment allocation for three consecutive days before ICH, and then tested on days 3, 14 and 21 after ICH . We obseMRI was used to measure the volume of the residual hematoma, which was similar between the groups . However, we have used a slightly larger sample size n = 11–14) than in our previous study (n = 9–10) –14 than  to 31.17Taken together, our current and previous data  indicateIntracerebral hemorrhage (ICH) is a common cause of neurological morbidity.Mesenchymal stromal cells (MSC)-based therapies have been studied as a promising alternative for treating ICH due to their potential to modify multiple pathways associated with brain damage and neurological recovery.We have previously shown that a single iv. injection of human umbilical cord Wharton’s jelly-derived MSC (hUC-MSC) 24 h after the induction of ICH reduced the residual hematoma volume in a model of moderate ICH but was inefficient when a severe ICH was modeled in rats.Considering that ICH is a medical emergency with rapidly progressing symptoms, we hypothesized that an earlier treatment could be more efficient in severe ICH.Here, we show that the hyperacute administration of hUC-MSC did not change the survival rate in a model of collagenase-induced severe ICH in rats.Motor function was not improved by the hyperacute hUC-MSC treatment.Hyperacute hUC-MSC transplantation did not altered the residual hematoma volume after a severe ICH.These results indicate the lack of efficacy of the iv. administration of hUC-MSC in the hyperacute phase of severe ICH.


Mental health problems currently affect a quarter of the world's population. Recent research in western societies has started to examine the relationship between entrepreneurship and mental health problems such as Attention Deficit Hyperactivity Disorder (ADHD) and dyslexia. However, little has been done to categorize entrepreneurs into different types and investigate how their levels of mental health and well-being correspond to these types. This study divided entrepreneurs into established and nascent categories and examined this topic in Singapore. By distributing two sets of surveys, a total of 834 responses were collected, with 346 responses from established entrepreneurs and 488 responses from nascent ones. The results showed that the nascent entrepreneurs' levels of well-being were found to be much lower than those of the established entrepreneurs. Furthermore, entrepreneurs with ADHD or dyslexia symptoms generally had a much lower level of life satisfaction, compared with those without. However, the self-care behaviours observed in this sample differed somewhat from observations made in western societies, which might be explained by the different cultures and habits in Singaporean society. The findings not only highlight the need for relevant organizations to support nascent entrepreneurs but serve to remind veteran entrepreneurs to practice more healthy self-care behaviours. In recent decades, as affluence levels have significantly improved and basic survival needs are no longer a concern, greater attention has been focused on people's spiritual needs and mental health. Mental health relates to people's emotional and behavioural well-being. It could have significant impacts on their daily life, work performance and relationships with others  was used as a screening test to identify respondents who potentially have ADHD. It was adopted from Kessler et al.  and thisThe Adult Dyslexia Checklist proposed by Vinegard  was usedFor self-care behaviours, respondents were asked to select how frequently they had performed each activity/item in a week, with 1 = Never, 2 = 1 time, 3 = 2 to 3 times, 4 = 4 to 5 times and 5 = 6 to 7 times. Some self-care behaviours were considered healthy, including getting enough sleep, exercise, yoga and meditation, eating healthy food and finding comfort in religion; some were considered unhealthy, such as smoking and drinking alcohol , it could be expected that this group of young entrepreneurs in Singapore would refrain from smoking and drinking in order to maintain a relatively healthier lifestyle.Next, a comparison was made between entrepreneurs with symptoms of mental health problems and those without. Generally, entrepreneurs with ADHD symptoms get enough sleep less frequently than those without ADHD. It was unexpected that the results for exercise and healthy food were almost identical for the two groups, while those with ADHD actually engaged in yoga, meditation and religious behaviours more often than those without ADHD. Concerning the two negative behaviours, those with ADHD displayed a significantly higher frequency of smoking and drinking compared to those without, and the same pattern was also observed for dyslexia. This corresponded to other research findings, which have reported that people with ADHD tend to have more problems sleeping and are more likely to smoke and drink a lot  and 15–20% are likely to have dyslexia  and with the 1964 Helsinki Declaration and its later amendments or comparable Ethical Standards. The Ethics Committee waived the requirement of written informed consent for participation.JG and ZX designed the guideline of the articles. SW, PO, and ZX contributed to the drafting of material for individual section. MG compiled the writing and conducted the analysis. JG and SW aligned the paper. YL and PO reviewed and provided corrections on the original draft. All authors contributed to the article and approved the submitted version.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.