Attention deficit/hyperactivity disorders (ADHD) and autism spectrum disorders (ASD) frequently co-occur. However, we know little about the neural basis of the overlaps and distinctions between these disorders, particularly in young adulthood – a critical time window for brain plasticity across executive and socioemotional domains. Here, we systematically review 75 articles investigating ADHD and ASD in young adult samples (mean ages 16–26) using cognitive tasks, with neural activity concurrently measured via electroencephalography (EEG) – the most accessible neuroimaging technology. The majority of studies focused on event-related potentials (ERPs), with some beginning to capitalise on oscillatory approaches. Overlapping and specific profiles for ASD and ADHD were found mainly for four neurocognitive domains: attention processing, performance monitoring, face processing and sensory processing. No studies in this age group directly compared both disorders or considered dual diagnosis with both disorders. Moving forward, understanding of ADHD, ASD and their overlap in young adulthood would benefit from an increased focus on cross-disorder comparisons, using similar paradigms and in well-powered samples and longitudinal cohorts. There is currently an intensive search for the neural basis of these disorders. Electroencephalography, or EEG, has emerged as an ideal cognitive neuroscience tool in developmental psychopathology research, as it is relatively non-invasive, inexpensive and accessible compared to other neuroimaging technologies . To date1.1ADHD is characterised by severe deficits in attention, hyperactivity and impulsivity, whereas ASD is associated with impaired communication and social interaction skills, in addition to repetitive and restricted behaviour and interests . These tThe phenotypic overlap between ADHD and ASD appears to be explained by aetiological overlap, at least partly, in terms of shared genetic influences between traits of both disorders. For example, individuals with ADHD and their siblings display more ASD symptoms than non-sibling controls, suggesting shared familiality . Twin stShared etiology between ADHD and ASD also points towards shared neurocognitive pathways. However, these disorders have, to date, been investigated in divergent fields. One reason is that dual diagnosis of ADHD and ASD was not formally permitted until the revision of Diagnostic and Statistical Manual of Mental Disorders in 2013 or DSM–5 . However1.2young adulthood or emerging adulthood  refers to transient time-locked EEG activity typically averaged across trials. Broadly speaking, early ERP components (100–200 ms) are thought to index stimulus-driven sensory processing, whereas later components are thought to index cognitive processing . In the EEG-imaging has been extensively used to investigate neural mechanisms underlying ADHD and ASD, but to date most of this work has been in children and adolescents. Broadly speaking, ERP research has highlighted atypical ADHD-linked cognitive profiles in inhibitory control and performance monitoring  and atyp1.4Our main goal is to systematically review studies using EEG-imaging in young adults with ADHD and/or ASD, focusing on both ERPs and EEG oscillatory measures. We aim to synthesize the numerous neurocognitive domains considered for each disorder independently, as well as when both disorders were directly compared and/or dual-diagnosis was considered. We include research focusing on diagnostic categories and traits to understand the mechanisms underlying these disorders more broadly. Traits and diagnosis appear to share common genetic origins, with diagnoses aetiologically represented as extreme ends of a continuum of behavioural traits . The curOur review is restricted to EEG recorded during cognitive tasks, which allows for the interpretation of brain activity in the context of putative cognitive operations being carried out. EEG during cognitive tasks is more reliable  while thOther imaging modalities are also beyond our current scope, but readers can refer to relevant reviews on the use of methods such as functional magnetic resonance imaging (fMRI)  and posi22.1Initial literature search and selection of studies were informed by guides from PRISMA  . Searche2.2Inclusion criteria were 1) empirical research published in peer-reviewed journals; 2) involving human participants; 3) EEG was elicited within a cognitive task; 4) the study examined case-control differences between individuals with ADHD/ASD as defined by either research/clinical diagnosis or trait measures, and/or examined traits of ADHD/ASD-traits as continuous variables; 5) mean age was between ages 16–26 (or fell within the age range reported in a study and the study focused on adults). Exclusion criteria were if the study 1) did not use EEG-imaging, 2) focused on resting states, neurofeedback, sleep states, and/or epilepsy/seizures and/or 3) had a sample size of fewer than 12 participants with ADHD/ASD.2.3Out of 3738 articles identified in the initial search , 3608 were discarded after screening and 75 articles were included in the current review (35 for ADHD and 40 for ASD). To organise this vast literature, the relevant studies are grouped according to the primary neurocognitive domain investigated, broadly defined as: 1) attention processing, 2) inhibitory control, 3) performance monitoring, 4) face processing, 5) imitation and empathy, 6) sensory processing, and 7) memory and language.Each of the neurocognitive domains is subsequently divided according to the focus on ADHD or ASD. ERP studies are described first, followed by those including data relevant to brain oscillations . We did not find any research in the relevant age period directly comparing both disorders or considering dual-diagnosis. This points towards a critical gap in the literature for ADHD and ASD research in young adulthood – we return to this issue in our discussion, where we highlight the candidate neurocognitive processes that are likely to inform research investigating the ADHD-ASD overlap and/or distinction in young adulthood.Details on the most prominent ERP and EEG oscillatory measures (qEEG and time-frequency approaches) discussed in this review, and their putative functional significance and/or definition, can found in Primary findings are summarised in terms of either significant differences between ADHD/ASD and non-ADHD/ASD control participants specific to the study, unless otherwise specified , and/or significant associations between ADHD/ASD traits and ERP/EEG measures. These are then interpreted, whenever possible, based on functional significance for neurocognitive processing. More details on the sample composition of each study can be found in subsequent tables, which also provide details on age (mean and range) and sample size of the ADHD/ASD group, information about the comparison group(s), cognitive paradigms employed, and ERP/EEG measures investigated. When multiple cognitive domains are relevant, studies are presented only under the primary category in the subsequent tables.33.1Sixteen articles investigated attention processing, including indices of attentional orienting, subsequent attentional allocation and sustained attention. All except two studies considered clinical or research diagnosis. Thirteen of the studies focused on ADHD and three focused on ASD. The most widely used cognitive paradigms to study attentional processing in ADHD were visual and auditory oddball paradigms, continuous performance task (CPT) and, to a more limited extent, a four-choice reaction time task called the fast task see .Table 2A3.1.13.1.1.1Several ERP studies have examined selective attention using oddball paradigms. In such studies, participants are typically presented with a sequence of standard auditory/visual stimuli intercepted with additional targets appearing less frequently. The Minnesota Twin Family Study  examinedTwo additional oddball studies in adults aged 23–24 years included two types of nontargets: constant nontargets, for which the same nontarget stimulus was repeatedly presented, and novel nontargets, for which a completely novel stimulus was presented each time . One study found that higher number of ADHD symptoms in a nonclinical sample were associated with more pronounced P3b amplitude for nontargets relative to targets, but only when constant nontargets rather than novel nontargets were considered . This suThe cued CPT  is a variant of the CPT task that is used widely in ADHD research to index aspects of attentional orienting, response preparation and response inhibition e.g., . In thisThe cued CPT has also been used to tease apart potential markers of persistence or remission of ADHD diagnosis or parental reports of ADHD symptoms at ages 18–19 with an earlier diagnosis of childhood ADHD . ADHD peTwo ERP studies have used the fast task . The tasIn one study, ADHD at age 18 was associated with reduced target-P3b amplitude in the baseline condition of the fast task, which in turn was also linked to higher RT variability (RTV), both of which suggest inefficient and inconsistent attentional allocation to targets . In addi3.1.1.2One study assessing EEG power  across tTwo studies using oscillatory approaches in tasks relevant to attentional processing focused on very-low frequency (VLF) oscillations (0.02-0.20 Hz) . It has 3.1.23.1.2.1Selective attention in ASD samples has also employed oddball ERP paradigms. One study using an auditory oddball paradigm suggests that ASD is associated with atypical perceptual processing, particularly increased perceptual capacity . In non-Another study using the visual oddball paradigm showed that ASD at age 17 was associated with both higher amplitude of frontal P3a and longer latencies of centro-parietal P3b to nontargets, which suggest that ASD is associated with atypical distractor processing . Similar3.1.2.2One study was identified which applied oscillatory approaches to studying attention processing in ASD. Event-related gamma activity  at age 16 were associated with reduced no-go-N2 amplitude , suggestAn auditory go/no-go paradigm also showed that ADHD at 25 was associated with reduced no-go-P3b amplitude and delayed no-go-N2 and no-go-P3b . Unexpecinhibit response to the target. The pattern of findings was slightly different when the instruction was instead to respond to the target: all subtypes also showed an attenuated target-P3a amplitude, with the exception of those with the combined subtype  for no-go trials, thought to index reduced response preparation  at ages 23–24 in a go/no-go task . Insteadparation   typically consist of congruent trials, where flankers and targets are compatible, and incongruent trials, where flankers and targets are incompatible. ERPs associated with this task include the N2, which is increased in incongruent trials and is thus thought to index ‘conflict monitoring’, or processing of high conflict stimuli that induce potential competing responses . Two addTwo studies employed flanker tasks in young adult samples with ADHD. Using an arrow flanker task , a studyError processing has also been investigated using a go/no-go paradigm, in which participants are typically required to respond to one type of stimuli  but withhold response to another type . For instance, ADHD at age 16  was assoAdditional studies based on ADHD traits investigated performance monitoring in the context of feedback and reward. In one study, participants were asked to respond to a target stimulus under two conditions: either a fast response would lead to both positive feedback and immediate start of the next trial avoiding a delay (escape condition), or instead a fast response would not influence the delay of the next trial at all (no-escape condition). Higher number of ADHD symptoms at age 21 was associated with larger differences in CNV amplitude between the escape versus no-escape conditions, which could be interpreted as reflecting greater response preparation to conditions in which delayed rewards could be avoided .Reward processing was also investigated in a large sample from an epidemiological longitudinal cohort at age 24 . In a mo3.3.1.2A study described previously using a go/no-go task  also exa3.3.23.3.2.1Performance monitoring in ASD has been examined using different tasks from those employed in the ADHD literature. For instance, one study examined neural response to different types of feedback: social , non-social (candy) and neutral (shape), using a paradigm in which participants were asked to seek positive feedback or avoid negative feedback . IndividA key aspect of monitoring performance is the ability to make predictions in uncertain environments. One preliminary study examined predictive processing using a visual target detection task in ASD at age 21 . Both AS3.3.2.2before predictive targets compared to random targets; however, this decrease was not found for ASD at age 21, suggesting a motor anticipation failure. This deficit was accompanied by a reduced benefit in RTs to predictable targets in individuals with ASD. In contrast, ASD was associated with increased alpha activity over frontocentral regions before the onset of predictable targets compared to random targets. Taken such findings together, ASD appears to be associated with a profile of reduced motor preparation to predictable targets, as reflected by findings on mu oscillations, alongside compensatory strategies to counteract such lack of motor preparation, reflected by findings on frontocentral alpha oscillations.The study above also examined the relevance of mu rhythms in prediction processing . Mu rhyt3.4Thirteen studies investigated face processing using EEG-imaging. Only two studies did not consider clinical or research diagnosis. Whereas two studies focused on ADHD, 11 focused on ASD. The cognitive paradigms employed examined various aspects of face processing, including face orientation, gaze direction, mouth shape, and comparisons to non-face stimuli and verbal stimuli of names. One of the studies in ASD focused on name processing only, but is included in this section due to its relevance to potential shared mechanisms with face processing see .Table 2D3.4.13.4.1.1only angry faces did not find the same N170 effects, but instead showed that ADHD at age 24 was associated with enhanced target-P3b amplitude and reduced target-N3 amplitude  in response to angry faces, which was interpreted as an index of more effortful processing  compared to incongruent face-voice pairings , whilst such a congruency effect was not observed in ASD at age 22 .Atypical patterns of ERP findings in relation to face processing in ASD, however, are not always found in young adult samples. One study failed to find group differences between ASD at age 23 and non-ASD controls in ERPs locked to face stimuli, such as P2 and N250 in response to familiar versus unfamiliar faces, and N400 in response to repeated versus novel faces . It is pASD-linked atypical profiles of N170, however, are not always consistent. For example, a study using house and face stimuli failed to replicate group differences in N170 between ASD and non-ASD controls at age 22 . AnotherTwo studies investigated the neural correlates of both name and face processing in ASD. One study compared attention to self-related stimuli (own + close other’s names and faces) to control stimuli (famous + unknown names and faces) . IndividOne study investigated the effect of gaze orienting on emotional facial expressions. The paradigm involved initial presentation of an individual’s face with neutral expression and with straight gaze ; the gaze then changes to either a right- or leftward gaze , followed by the same individual with an either happy or fearful expression . Unlike 3.4.2.2Name processing has also been examined in relation to brain connectivity , which w3.5Six articles investigated processing relevant to behavioural imitation and empathy. All of these studies focused on ASD only, and considered clinical or research diagnosis. The focus on ASD may be driven by theoretical perspectives linking deficits in imitation with aspects of social cognition that are thought to be ‘disrupted’ in ASD, such as difficulties with empathy e.g., . Cogniti3.5.1No studies were identified which investigated imitation and empathy in ADHD in young adults using EEG-imaging, using either ERPs or oscillatory approaches.3.5.23.5.2.1One study used ERPs to investigate the perception of pain experienced by others , and fouFurther support for atypical early pain processing in ASD at age 20 was demonstrated by a reduced P2 amplitude induced by thermal pain, administered with a contact heat-evoked potential stimulator on the right lateral leg, despite equivalent self-reported pain levels to non-ASD controls . Adminis3.5.2.2A series of studies has examined the mu rhythm in ASD. Mu suppression has been proposed to reflect activity in the human mirror neuron system  Table 1, althougexecution; however, contrary to non-ASD controls, reduced mu suppression during hand movement observation was found for ASD at age 23 . The majority of studies on ASD reflects the widely reported hyper- and hypo-sensitivities in sensory processing in ASD which has been recognised as an important feature of ASD in the latest DSM . Paradigms include those that examine low-level stimulus characteristics such as orientation and brightness, as well as those using more socially-relevant stimuli such as vocal sounds and faces see .Table 2F3.6.13.6.1.1ADHD has been associated with atypical profiles of sensory processing across various modalities. In a study examining colour perception, ADHD at age 16 was associated with an enhanced P1 amplitude to blue-yellow chromatic stimuli, but typical P1 amplitude for red-green stimuli and typical performance on an ophthalmological exam . P1-relaAtypical auditory processing has also been identified in ADHD, using a sound perception task. For this task, while watching a silent video, trains of consecutive 5-tones were presented (with onset-to-onset time of 400 ms), separated by either 1 s or 5 s between each train . ADHD inIn addition to traditional scalp-based data, one study also used data derived from independent component analysis (ICA), a statistical technique that can separate neural and non-neural sources in the EEG signal thus leading to improved signal-to-noise ratio and source estimation . ICA was3.6.1.2The same study focusing on trial-to-trial variability of early-sensory components  also exa3.6.23.6.2.1In the auditory domain, discrimination of spoken syllables of varying emotional prosodies were investigated using a paradigm that elicited mismatch negativity (MMN), an ERP component thought to index detection of unexpected stimuli  Table 1. ASD at Some studies investigated visual processing exclusively. Using a discrimination task (of stimuli made of black line segments), non-ASD controls showed a negative ERP deflection around 120 ms post-stimulus, believed to signal boundary detection . This effect was diminished in ASD at age 20 . AnotherAdditional experiments used visual evoked potentials (VEP), such as P1 and N80, to investigate integrity of the visual pathway: ASD was associated with reduced P1 amplitude in a pattern-reversal paradigm at age 21 ; and alsOther studies investigated the integration of different modalities. One study found that both ASD at age 21 and non-ASD controls showed equivalent reductions in amplitude of N1 and reductions in latencies of N1 and P2 in response to audio-visual stimuli relative to unimodal stimuli . In contSensory processing in ASD has also been examined in interpersonal contexts. A study examined neural responses to stimuli representing various levels of interpersonal distance preferences to strangers or friends . For ASD3.6.2.2Sensory processing in ASD has been studied in non-social domains using oscillatory measures. Two studies showed patterns of atypical context modulation during early visual perception in ASD. In one of these, participants were presented with Gabor patches with different degrees of orientation . It was 3.7Seven articles investigated various processes related to memory, learning and language. Five of the studies considered clinical or research diagnosis. Three of these focused on ADHD and four on ASD. A variety of cognitive paradigms were investigated, which examined short-term or working memory (WM), long-term episodic memory and semantic memory/language processing see .Table 2G3.7.13.7.1.1Two studies examined WM, which is the ability to temporarily hold and manipulate information in mind. In a delayed match-to-sample task, participants were asked to judge whether target stimuli (abstract figures) were presented in previous trials . In addi3.7.1.2A study analysed event-related oscillatory activity in the maintenance phase of the delayed match-to-sample task described previously to investigate WM , that is3.7.23.7.2.1One study investigated long-term memory of visually-presented words, and found that despite equivalent memory performance, ASD at 25 was associated with differential topographical neural activity patterns during recognition memory of old versus new items: in the parietal instead of anterior regions within 300–500 ms post-stimulus, and attenuated at the right frontal regions within 800–1500 ms post-stimulus . Such a Another aspect investigated in ASD is language processing, using paradigms eliciting N400 – a putative marker of effort for processing and integrating meaning  Table 1. For exa3.7.2.2A study investigated gamma and theta oscillations during speech processing in ASD at age 20 . While n4We set to conduct a systematic review of EEG-imaging studies (ERP and EEG oscillations) with ADHD and/or ASD. The EEG literature in these disorders is vast and has rapidly expanded since the early 2000’s, as revealed by the number of studies we have found despite restricting the sample to young adults. The studies were highly variable: the majority used varying participant samples, diagnostic procedures, and EEG analytical approaches . We wereWhat is most important now, we believe, is the need to consolidate this ever-evolving but disparate literature, and call for the fields of ADHD, ASD and EEG to come together with the common goal of understanding the overlap and distinction between the two disorders. Below we provide a synthesis of the candidate disorder-overlapping and disorder-specific neurophysiological markers of ADHD and ASD, as informed by the most up-to-date and comprehensive literature on EEG-imaging in young adult samples.4.1We intend to draw broad functional patterns across neurocognitive domains and paradigms in this general discussion. An overview of the putative cognitive-neurophysiological basis of ADHD and ASD, as well as their overlap and distinction, is presented in Both ADHD and ASD have been linked to atypical allocation of attentional resources and atypical performance monitoring, but there are also atypical processing aspects that are disorder-specific. In ADHD, much atypical processing in the attentional domain relates to orienting to cues that signal response preparation, whereas in ASD it appears to be more specific to novelty, an effect that may be at least partially influenced by increased perceptual capacity . ADHD may relate to atypical monitoring of conflict and of rewarding feedback, whereas ASD may relate to atypical feedback processing involving socially-relevant stimuli. Additionally, ADHD has also been linked to diminished inhibitory control, which has not been directly studied in ASD during young adulthood.Both ADHD and ASD have also been linked to atypical processing of faces, and atypical early sensory processing in both visual and auditory domains. However, disorder-specific profiles may qualify each disorder. In ASD, the evidence for atypical face processing is mixed, at least in young adulthood and may depend on the self-relevance quality of the stimuli, whereas in ADHD the atypical processing may be specific to anger emotions. For ASD, atypical sensory processing has been suggested by various studies to contribute to ‘later’ cognitive deficits such as face processing and attentional processing. Some domains have been studied exclusively in ASD, thus direct comparisons with ADHD are required, including evidence of atypical language/semantic processing and mixed findings on atypical imitation processing.4.2A critical gap in the literature is investigation across disorders. Some studies have compared either ADHD with other clinical groups , and ASDThe other major limitation of the literature is the overly large sampling interval used in some studies with young adult samples, despite the mean age falling within our specified cut-off (ages 16–26) (Tables 2A–2G). Some of the studies, particular in the ASD literature, included a broad age range (overlapping with children and late adult samples) even if the mean age fell within our pre-specified period. The brain undergoes changes across the lifespan even after childhood and adolescent . A trulyOne important issue in relation to age is that the prefrontal cortex develops throughout adolescent until around the age of 25 , and thuA neglected issue is the role of the informant in biomarker research. This review revealed that, at least in ADHD, the reliability of neurophysiological indices across various cognitive domains, particularly those based on ERP components, will differ depending on whether group classifications are based on informants   rather tThe various research considerations described above would require large samples for appropriate statistical power, a common problem in neuroimaging studies. The majority of studies in our review involved small samples, typically recruited via convenient sampling methods , with a few exceptions e.g., . As youn4.34.3.1The review points towards opportunities to consider the design of task paradigms, assessing various neurocognitive domains, which are likely to inform our understanding of the ADHD-ASD co-occurrence and its neural basis. These designs, for example, may include experimental manipulation of task parameters building on the existing literature. We will briefly go over some possibilities in relation to each domain.For attentional processing, the most common paradigm used across ADHD and ASD is variations on the oddball paradigm. Thus, one may consider task parameters that could reveal disorder-specific atypical processing. For instance, ADHD-specific profiles may depend on stimuli modality  and ASD-related atypical attentional processing may be modulated by task load  and/or sTask paradigms that are broadly relevant to socioemotional processing might also combine parameters that have been considered separately in the ADHD and ASD literature. Face processing tasks could, for example, consider a range of positive and negative emotions, so to establish if processing of specific emotions are disorder-specific e.g., , 2015b. Finally, atypical sensory processing across auditory and visual domains have been reported in both ADHD and ASD. One issue is the heterogeneity in stimuli employed, spanning colour , orientaAn advantage of including additional task conditions is the improvement in functional interpretation of dimensions of neurocognitive processing to each disorder. However, we acknowledge that it might not always be practical/viable to include such conditions, for example, because of the additional burden to the participant during data collection . Therefore, task parameters should be considered on a case-by-case basis depending on the main research aim, the resources available to the study, and the nature of the participant sample. One could consider smaller-scale studies to establish functional interpretation for paradigms for which findings are more controversial, in combination with larger-scale studies using more established paradigms for whch findings have more broadly-accepted functional interpretations.4.3.2We also suggest further research considerations to better understand the neural basis of ADHD and ASD (and their co-occurrence) within a developmental psychopathology framework. For example, genetically-informed studies can be used to clarify the etiology of the overlap and distinction between ADHD and ASD, by helping tease apart the genetic and environmental contributions to these disorders and their co-occurrence in young adulthood. Family studies or twin studies, in combination with EEG-imaging, could be used to estimate whether the neurocognitive profiles associated with ADHD and ASD stem from common or distinct aetiologies. Such study designs could shed lights on the neurocognitive mechanisms underlying the shared genetic influences between the disorders .Additional longitudinal research, both within and across ADHD and ASD, is also required to track changes in neurophysiological processes from childhood into adulthood. Longitudinal studies are particularly relevant in light of persistence of symptoms despite a drop in diagnosis e.g., , potentiFinally, given the higher incidence of mental health risk in ADHD and ASD , future 4.3.3Recent hardware developments in mobile EEG have led to new portable, light-weight and increasingly affordable EEG systems. Mobile EEG technologies might constitute an ideal tool to ease data collection from large number of participants within a relatively short timeframe, and within powerful developmentally-informative research design , such asEEG-imaging research is currently exploiting recent advances in signal processing, which are likely provide additional information compared to traditional analytical approaches. Such ‘microscopic’ examination, exploiting all available EEG data, provides novel opportunities to better link the underlying neurophysiology of these disorders with their cognitive and behavioural profiles, as well as with their overlap and distinction. Among these approaches, ICA can be used to separate neural from non-neural activity, increasing the signal-to-noise ratio of the neural components , thus ai4.4We have focused exclusively on EEG-imaging, but other imaging modalities such as fMRI or MEG, which are typically thought as having better spatial resolution, can also contribute to research into the neural basis of the ADHD-ASD overlap and distinction . The useOur review consists of a qualitative rather than a quantitative synthesis of the available evidence. While we have identified a relatively high number of studies in young adult samples (75 articles), these were spread across neurocognitive domains and have employed heterogeneous cognitive paradigms – precluding pooling of the data across studies at this stage, for instance, via meta-analyses. Here, we highlight the candidate neurocognitive processes and markers leveraging EEG-imaging, alongside established and novel cognitive paradigms, which could provide the foundation for the next phase of research in ADHD-ASD overlap. We also seek to galvanise joint collaborative efforts, such as data-sharing, so that a more formal quantitative reviews could be conducted in the near future. As the literature in this age group accumulates, further reviews could focus on specific age ranges and specific neurocognitive processes/tasks within this developmental period.Despite reviewing a relatively large number of studies, we may have missed out on important studies that are nevertheless relevant to our aim of understanding the overlap between ADHD and ASD. For example, intra-participant variability in sensory processing (as assessed by ITC) was discussed in the context of ADHD Section  but not 5EEG-imaging studies in ADHD and ASD are rapidly accumulating, reflecting the field’s enthusiasm to leverage EEG methods, and the potential of this research to inform the neural basis of the ADHD-ASD overlap and distinction. Despite the lack of studies directly comparing both disorders or including dual diagnosis, some consistent candidate disorder-overlapping and disorder-specific biomarkers have emerged. Moving forward, research into overlap and distinction that harnesses EEG-imaging should focus on cross-disorder comparisons using the similar paradigms, better powered studies and larger samples, more restricted age ranges, well-characterised longitudinal cohorts, and further applications of time-frequency and source-based EEG approaches.


The optimal insertion angle for suture anchor insertion has long been of great interest. Although greater tuberosity decortication is commonly performed during rotator cuff repair, the effect of decortication on the suture anchor insertion angle remains unclear. The purpose of this study was to compare the pullout strength of threaded suture anchors inserted at 45° and 90° in decorticated and non-decorticated synthetic bone models.Two kinds of synthetic bones were used to simulate the decorticated and non-decorticated conditions, for which 40 metallic suture anchors were used. Anchors were inserted at 45° and 90° in both decorticated and non-decorticated models and tested under cyclic loading followed by load-to-failure testing. The number of completed cycles, ultimate failure load, and failure modes was recorded.p <  0.001). In the non-decorticated model, the ultimate failure load of anchors inserted at 45° (591.8 ± 58 N) was also significantly lower than that of anchors inserted at 90° (724.9 ± 94 N) (p = 0.003). Due to the diverse failure modes in the non-decorticated model, specimens with a failure mode of suture anchor pullout were analyzed in greater detail, with results showing a significantly larger pullout strength for anchors inserted at 90° (781.6 ± 53 N) than anchors inserted at 45° (648.0 ± 43 N) (p = 0.025).In the decorticated model, the ultimate failure load of anchors inserted at 45° (67.5 ± 5.3 N) was significantly lower than that of anchors inserted at 90° (114.1 ± 9.8 N) (Regardless of decortication, the pullout strength of anchors inserted at 90° was greater than those inserted at 45°. The clinical relevance is that inserting suture anchors at 90° is recommended due to the significantly larger ultimate failure load in both decorticated and non-decorticated bones. Techniques involving suture anchors have become prevalent for securing soft tissues to the bone in different parts of the body –5, the mGreater tuberosity (GT) decortication is commonly performed during rotator cuff repair since it may promote biological healing at the bone-tendon junction . AlthougDespite the insertion angle of suture anchors having been discussed in various biomechanical models , 15–19, 3 was cut into blocks measuring 60 mm in width, 42 mm in depth, and 40 mm in height (named decorticated synthetic bone). To simulate the non-decorticated bone, blocks of the same polyurethane foam were laminated with 2 mm of short-fiber-filled epoxy (density of 1.63 g/cm3) attached on one side (named non-decorticated synthetic bone). Tingart et al. reported that the volumetric bone mineral density of the humeral GT ranged from 0.10 ± 0.03 to 0.18 ± 0.04 g/cm3 in a cadaveric study [3 was selected. In addition, the 2-mm-thick cortical bone was chosen in accordance with a previous study [Two kinds of synthetic bones  were used in this study. To simulate the decorticated bone, solid rigid polyurethane foam with a density of 0.16 g/cmic study ; accordius study .Metallic suture anchors  with double-loaded no. 2 braided sutures were used for biomechanical testing. As suggested in previous studies , 27, theEach synthetic bone block was mounted on a universal material testing system . A custom-made clamp was used to fix the synthetic bone block, after which the suture ends were knotted together and looped over a post on the adapter of the material testing machine. The custom-made clamp was rotated to make a 45° angle between the suture and the bone surface Fig. . SuturesSimilar to previous studies , 22, eacU test was used to compare the ultimate loads to failure for the suture anchors at 45° and 90° in both the decorticated and non-decorticated groups. Statistical significance was set as a p value less than 0.05.Statistical comparisons were performed with SPSS for Windows, Version 16 . Descriptive statistics, including means and standard deviations, were obtained for each group. The Mann-Whitney Post-hoc power analyses were performed with G*Power Version 3.1.3  to calculate the achieved power. An alpha equal to 0.05 was given for these models.p <  0.001). The calculated effect size of the results was 5.94; and with a given α equal to 0.05, the post hoc achieved power was 99.9%. All specimens in the D-45 and D-90 groups failed from suture anchor pullout  was significantly smaller than that in the D-90 group (114.1 ± 9.8 N) (p = 0.003). The calculated effect size of the results was 1.70, and with a given α equal to 0.05, the post hoc achieved power was 93.8%. The failure modes in both groups varied. In the N-45 group, three specimens failed from suture anchor pullout, two failed from suture rupture at the eyelet, and five failed from a broken eyelet. In the N-90 group, five specimens failed from suture anchor pullout and the other five failed from suture rupture at the eyelet  was significantly smaller than that in the N-90 group (724.9 ± 94 N) (.8 ± 58 Np = 0.025). The calculated effect size for these data was 2.67, and with a given α equal to 0.05, the post hoc achieved power was 83.4%.Due to the diverse failure modes in the non-decorticated bone model, specimens with a failure mode of suture anchor pullout (3 in the N-45 group and 5 in the N-90 group) were analyzed in greater detail. The ultimate failure load of these specimens in the N-45 group (648 ± 43 N) was significantly smaller than that of the specimens in the N-90 group (782 ± 53 N)  were much higher than those in decorticated bones (68 N and 114 N for insertion angle of 45° and 90° respectively). Thus, in clinical practice, surgeons should also pay attention to the effect of decortication.Although previous studies have evaluated the pullout strength of suture anchors in a model with synthetic bone blocks that had similar properties to cancellous bone , 15, theThe current study demonstrated that the ultimate load to failure was significantly higher when the suture anchors were inserted at 90° compared to 45° in the non-decorticated synthetic bone model, which is consistent with previous research . NagamotIn addition to biomechanical studies, the optimum insertion angle of a suture anchor has also been intensively discussed. Dr. Burkhart has addressed several comments on this topic, with one of the flaws pointed out being that the eyelets of the suture anchors were not at or below the surface level of the bone . In respIt is worth noting that the optimal insertion angles for threaded and threadless anchor were different. Both the previous study  and our Although anchor pullout was the most common failure load in our study, it may not occur often in non-decorticated bones clinically since the suture materials were replaced. According to the previous studies , 30 and There are some limitations in this study. First, the current study used synthetic bones for biomechanical testing, rather than cadaveric humeri. Nevertheless, this biomechanical model has been applied in several studies , 7, 15. Regardless of decortication, the pullout strength of anchors inserted at 90° was greater than those inserted at 45°.


Individuals with Attention-Deficit Hyperactivity Disorder (ADHD) are often characterized by deficits in working memory (WM), which manifest in academic, professional, and mental health difficulties. To better understand the underlying mechanisms of these presumed WM deficits, we compared adults with ADHD to their peers on behavioral and neural indices of WM. We used a visuospatial change detection task with distractors which was designed to assess the brain’s ability to effectively filter out distractors from WM, in addition to testing for effects of WM load. Twenty-seven unmedicated adults with ADHD were compared to 27 matched peers on event-related potential (ERP) measures of WM, i.e., the contralateral delay activity (CDA). Despite severe impairments in everyday life functioning, findings showed no difference in deficits in behavioral tests of working memory for adults with ADHD compared to their peers. Interestingly, there were differences in neural activity between individuals with ADHD and their peers showing that the CDA of individuals with ADHD did not distinguish between high, distractor, and low memory load conditions. These data suggest, in the face of comparable behavioral performance, a difference in neural processing efficiency, wherein the brains of individuals with ADHD may not be as selective in the allocation of neural resources to perform a WM task. Attention-Deficit/Hyperactivity Disorder (ADHD) is a prevalent mental health disorder characterized by persistent behavioral symptoms of inattention and hyperactivity/impulsivity, which can severely impede educational, occupational and health outcomes . VisuospWM refers to a limited-capacity, multi-component cognitive system that is required to keep relevant information in mind for a few seconds while performing complex tasks such as reasoning and comprehension . EvidencIn this study, we examined neural indices of storage capacity and filtering efficiency of adults with ADHD and their peers using a visuospatial change detection working memory task while electroencephalography (EEG) was recorded. Similar to The sensitivity of the CDA waveform to load can also be used to determine an index of filtering efficiency: in other words, whether irrelevant items unnecessarily consume WM capacity. In the distractor version of this task, participants are asked to remember the colors of squares (two squares in the low load and four in high load) in an array but to ignore the circles in a distractor condition (two squares + two circles). If the CDA of the distractor condition (two circles + two squares) resembles the low load condition (two squares), we can conclude that participants have effectively filtered out the irrelevant circles and excluded them from storage. Conversely, if the CDA for the distractor condition resembles that of the high load condition (4 squares), participants may have failed to efficiently filter out the distractors. Reviews of the neural origins of the CDA have concluded that the waveform is likely generated by multiple sources in the brain, of which the posterior parietal cortex appears most consistently mentioned . PosteriTo the best of our knowledge, only two studies thus far have investigated neural correlates of storage capacity using the CDA in adults with ADHD and their peers with inconsistent results. Given the paucity and conflicting nature of the research in this area to date, we tentatively formulated our hypotheses as follows: (1) the ADHD group, when compared to the comparison group, will show lower CDA-Δ, as operationalized by a difference measure between the CDA of the low and high load condition. We expected this effect to be small since literature has shown visuospatial-WM impairments in ADHD to be neither ubiquitous nor large, especially in college students ; and (2)Data reported in this manuscript were taken from a larger study investigating changes in neural and behavioral indices after a working memory training program conducted at the University of Toronto from 2011 to 2013 . Participants received $20 for their participation. Informed written consent was obtained from all participants prior to beginning the assessments  measures, we administered the Cambridge Neuropsychological Testing Automated Battery (CANTAB). Participants in this task needed to remember the spatial sequence of squares that briefly flashed on the screen in the same order  or reverse order  they appeared . As an eOur Visual WM task was adapted from The task had three conditions: in the low memory load condition (LL), the memory array contained two colored squares in each hemifield; in the high memory load condition (HL), the memory array contained four colored squares. In the distraction condition (DL), the memory array contained two colored squares and two colored circles see also . Particiand not their eyes, to the array on the side they were instructed to attend to. Also, they were told to sit in a comfortable, relaxed position and minimize eye blinks, verbalizations, and movements as much as they could. During the practice block, research assistants were actively checking for eye-movements and would correct behavior until participants understood and complied when acting out instructions.Special care was taken with task instructions considering the nature of our study population. Instructions were presented on several subsequent screens and read aloud by a research assistant who would verify that the participant understood the instructions. For example, participants had to repeat verbally that they were to ignore the circles in the distractor condition. Participants were also told that it was important to keep their eyes focused on the fixation cross in the center of the screen for the duration of the trial and to direct their attention, Participants’ WM capacity (referred to as ‘K’) was calculated from their behavioral performance. Similar to SD = 15) trials for the High Load, 83 (SD = 14) trials for the Low Load, and 80 (SD = 15) trials for the Distractor Load. The ADHD group had 62 (SD = 15) trials for the High Load, 72 (SD = 17) trials for the Low Load, and 66 (SD = 17) trials for the Distractor Load. Trial count was not a significant factor when it was added as a covariate in the main analyses.EEG was recorded with a 128-channel Geodesic Hydrocel Sensor Net at a 500 Hz sampling rate, using EGI Netstation stand-alone software . Data processing was kept similar to previous literature reported on the CDA e.g., . EGI’s NAfter average-referencing, data were transferred to Matlab 9.1  for further processing. Lateral-posterior sites were chosen to calculate the CDA. This selection of electrodes was based on previous studies e.g.,  and inspCDA-Δ and filtering efficiency were two measures derived from the CDA waves. CDA-Δ was calculated as the difference, or increases, in amplitude between the CDA amplitudes for the low and high load condition  were computed to ascertain effect size. According to η2 = .01 corresponds to a small effect, partial η2 = .10 corresponds to a medium effect, and partial η2 = .25 represents a large effect.In general, standard analysis of variance analysis (ANOVA) and t-tests along a moving window with 100 ms length and 50 ms overlap for the 300–850 ms duration of the retention period. Within a group, we tested for differences between the High and Low load CDA conditions. Between groups, we tested for differences in filtering efficiency and the CDA-Δ (the CDA-Δ was the H-L difference measure). Previous literature examining the CDA in ADHD reported different time windows for their analysis .F = 5.229, p = 0.027, η2 = 0.0947, with the comparison group showing a larger difference between the HL and LL compared to the ADHD group. No statistically significant group-difference effects were found for filtering efficiency at any time point . The results were computed separately for the Comparison and the ADHD group. Findings can be summarized by stating that none of the correlations between the neural measures  and our questionnaire  or behavioral performance measures  were statistically significant.The present study investigated whether and how adults with ADHD differ from their peers in aspects of working memory, such as CDA-Δ and filtering efficiency, as differentiated at a neural level using a visual change detection task with distractors. The ADHD group self-reported severe impairments on measures of everyday functioning and ADHD symptomatology. Despite being severely impaired, findings showed that the ADHD group did not differ from their peers on most behavioral indices of working memory performance nor in the neural measure of filtering efficiency. However, the data did reveal a reliable pattern of lower CDA-Δ  for the ADHD group.relatively high-functioning adults with ADHD, especially in their performance on standardized neuropsychological tests  are not involved, as our measure of filtering efficiency requires a separation between CDAs of the high and low load condition. Instead, our data showed perhaps a greater neural impairment in that the brains of individuals with ADHD did not differentiate between load conditions at all. This would suggest they were dedicating a similar amount of neural resources to the high and low load condition. Or perhaps individuals with ADHD have basic problems with encoding stimuli rapidly , which wn = 17) which may not have allowed for the detection of differences at a statistical level and, as Our CDA findings appear to differ from While it would be premature to characterize the CDA as an endophenotype of ADHD  and we dWe also want to point out limitations and alternative explanations/potential moderators of our effects. First, the task was not entirely self-paced  and different conditions were presented in a random manner mixed within blocks. Furthermore, there was a jitter period which also made the timing of the onset of the stimulus unpredictable. As such, it is possible the neural effects could also be attributed to difficulties with task switching and readiness or the timed allocation of attentional resources . Second,Notwithstanding the aforementioned limitations, the present study contributes to an emerging literature investigating working memory in subjects with ADHD from a neural perspective. Our findings suggest that the brains of individuals with ADHD process the maintenance of information differently from their healthy peers. More research would be needed to confirm our findings and further specify what this means for the characterization of this debilitating condition.10.7717/peerj.5601/supp-1Supplemental Information 1Click here for additional data file.


PGRP-LA, -LB, -LD, -LE, and -S1 during infection with two different fungal entomopathogenic strains. Furthermore, our data shows temporal differences in PGRP elicitation, with most PGRPs displaying significant upregulation at 60 h post-infection. Depletion of certain PGRPs via RNAi silencing resulted in a significant increase in fungal proliferation and a reduction in mosquito survival that was fungal strain-specific. Our data indicates that PGRPs play an important role in the antifungal response and expands our understanding of the factors that determine host susceptibility to fungal entomopathogens.Fungal entomopathogens are potential tools for the control of mosquito vectors that transmit infectious agents that cause disease in humans and animals. During the infection process, effective recognition of the invading fungi by the mosquito, is a crucial step in mounting an appropriate anti-fungal response. In this study, we investigated the role of peptidoglycan recognition receptors (PGRPs) in host resistance to fungal entomopathogens at the early stages of infection. Our study identified the induction of  Aedes aegypti, is one of the most important arboviral vectors, responsible for transmitting dengue, Zika and yellow fever virus , recent research has found that these recognition proteins play important roles against other invading microbes such as protozoan parasites Meister, . PGRPs cDrosophila and mosquitoes have shown that PGRP-LC plays a critical role in bacterial clearance and anti-Plasmodium defense  , are thought to function as negative regulators of immune responses given that its knock-down increased the production of several AMPs  colony was reared at 27°C and 80% humidity with a 12 h:12 h light/dark cycle. The colony was maintained by feeding adult females via an artificial feeding system using citrated bovine blood (HemoStat Laboratories Inc.). Adults were provided with a 10% sucrose solution and larvae were reared on a mixture of rabbit food and tropical fish food. Mosquitoes used in all experimental assays were conducted using 3- to 5-day old adult females.Beauveria bassiana (MBC 076) and Isaria javanica (ARSEF 5874) were grown on ¼ strength Sabouraud dextrose agar and yeast extract (SDAY) medium at 26°C for 15-days. Spore oil formulations were prepared as previously reported . The LT50 and LT95 values were calculated by probit analysis using SAS 9.4 statistical package.Fungal infection assays were conducted as previously described , six GNBPs (GNPS-1 through GNBP-6) and two molecules known to be elicited in response to fungal infection (CLSP2 and TEP22) was conducted in a 10 μl reaction using one microliter of the generated cDNA. The PowerUp SYBR green Master mix qPCR kit (Qiagen) was used in all reactions with gene specific primers .For gene expression analyses, pools of five challenged mosquitoes were collected at 48, 60, and 72 h post-infection (PI). These time points were selected to assess the progression of early immune elicitation when infected by these two different entomopathogenic fungal species. RNA was extracted from samples using TRIzol (Invitrogen) according to the manufacturer's instructions. Concentration and quality of the extracted RNA were evaluated via NanoDrop (Thermo Scientific). Synthesis of cDNA was conducted on normalized amounts of RNA using the QuantiTec reverse transcription kit with DNA Wipeout (Qiagen). Quantitative real-time PCR to amplify seven PGRPs ( primers . RT-qPCR primers . Rps17 hAe. aegypti. The HiScribe T7 Quick High Yield RNA synthesis kit (New England Biolabs) was used to generate dsRNA products from each target gene and each product was then diluted with molecular-grade water to a 3 μg/μl solution. Cold anesthetized 2- to 3-day old Ae. aegypti females were injected with 69 nl of a 3 μg/μl dsRNA solution into the thorax using a nano-injector . For infection assays evaluating the effect of silencing on mosquito survival, mosquitoes were challenged with entomopathogenic fungi at 1-day post-dsRNA injection. Silencing efficiency relative to dsFluc controls was evaluated for each silenced gene in mosquito whole-bodies at 3–4-days post-injection. We conducted a triple silencing of the three most highly expressed PGRPs , to overcome a potential redundancy in PGRP-derived protection. For the triple-knockdown, dsRNA products for each target gene were combined to a final concentration of 3 μg/μl each and with a final concentration of 9 μg/μl dsRNA injected per mosquito. To account for the total dsRNA product injected into dsPGRP-treated mosquitoes, control mosquitoes were also injected with 69 nl of a 9 μg/μl of dsFluc solution. Primers designed for gene silencing are reported in The transient depletion of PGRP genes was conducted via RNA interference (RNAi)-directed silencing as previously described . The error bars indicate the standard error of the mean and the type of test used is described in the respective figure legend.Graphs and statistical analysis for survival curves and gene expression were conducted using the software Prism 8 (GraphPad). Significance of survival curves was assessed using the Kaplan-Meier estimator with Log-rank test, while gene expression values were compared via ANOVA with Dunnett's post-test. LTAe. aegypti PGRPs can respond to entomopathogenic fungal infection, mosquitoes were challenged with either soybean oil, B. bassiana or I. javanica. Samples were collected at 48, 60, and 72 h post-infection (PI) and the expression of seven PGRPs    were evajavanica . In contPGRP-LC indicated absence of elicitation at 48 and 60 h PI but displayed significant increase in expression at 72 h PI only in mosquitoes infected with I. javanica  during the early time of infection. Our data shows that PGRP-S1 was the only PGRP that displayed consistently higher levels of gene expression throughout the time points of infection  . Silencing of the remaining PGRPs displayed similar phenotypes to that of dsPGRP-LA, with no significant effect on the survival of B. bassiana-infected mosquitoes in which PGRP-LD      . Interes = 0.39) , PGRP-LE = 0.45) , PGRP-S1 = 0.49) , or PGRP = 0.86) , had beeI. javanica-challenged mosquitoes showed a significant reduction in survival compared to dsFluc controls, with the depletion of either PGRP-LA        , PGRP-LD= 0.035) , or PGRP= 0.042) . No sign= 0.197) , PGRP-LE= 0.087) , or PGRP= 0.114) , Table 1PGRP-LA, PGRP-LD and PGRP-S1) and assessed its effect on mosquito survival. The silencing efficiency observed in tripled-silenced mosquitoes was the same as the single-silenced targets . Triple silencing prior to fungal infection led to a slim, albeit significant decrease in mosquito survival compared to the control groups in mosquitos infected with B. bassiana    , Table 1= 0.036) , Table 1B. bassiana-infected mosquitoes  are a family of proteins whose diverse functions, covering these three areas, are used to maintain the homeostasis between resistance and tolerance to microorganisms  and a later induction of GNBP-2 (at 60 h PI). These two GNBPs have been associated with the response to fungal infection , another family of proteins associated with fungal recognition. Here, our results indicate an earlier triggering of PGRP-LA, PGRP-LD, and PGRP-S1, had a robust and significant increase in gene expression in most time points evaluated. This was observed independent of the strain of fungal entomopathogen infecting the mosquito. In general, most of these PGRPs have been associated with the Imd pathway, and its triggering is thought to occur via the detection of peptidoglycan (PGN) during bacterial infections  at the same time of infection, providing further support that the activation of mosquito PGRPs is triggered by fungal-derived molecules.The PGRP ability to detect multiple pathogen-derived molecules might be inherent to their function and structural composition. Indeed, structural analysis of PGRP domains in Meister, . For insI. javanica but with no effect in those infected with B. bassiana. This detrimental effect of PGRP depletion was observed in three out of six PGRPs tested. This included PGRP-LA and PGRP-LD, which displayed strong gene elicitation during the infection process.We next corroborated the role of PGRPs in the antifungal defense by depleting PGRP expression via RNAi prior to fungal infection. Our results suggest that the strength of the PGRP anti-fungal response varies according to the invading fungal strain, with PGRP-silencing significantly affecting survival in mosquitoes infected with PGRP-SC2 depletion despite the absence of a significant induction in gene expression. This phenotype could be due to its predicted function as a PGRP with amidase activity, acting as a negative regulator of the immune response. Thus, while constitutively expressed, at basal levels it might be involved in catalyzing fungal-derived molecules that could potentially lead to a deleterious overstimulation of the immune response. In fact, a previous study in Drosophila, demonstrated that removal of PGRPs with amidase activity led to the uncontrolled activation of the Imd pathway and reduced fly survival due to an excessive immune response  hint to this possibility, with a slight but significant reduction in survival in mosquitoes infected with B. bassiana. Furthermore, PGRP expression analysis in single PGRP knockdowns indicated the significant elicitation of PGRP-LD in mosquitoes in which PGRP-LA or PGRP-LC were silenced. This suggests partial redundancy in these PGRPs and agrees with what is known with respect to PGRP-LA, PGRP-LD, and PGRP-LC, as interacting partners of the Imd pathway.The absence of any detrimental effect of PGRP silencing on survival of PGRP-LC-depleted mosquitoes challenged with B. bassiana but absent in I. javanica infection is puzzling. It could indicate that elicitation of PGRP-LC, and thus Imd pathway activation, is somewhat counterproductive to mosquito survival under the context of a fungal infection. However, our previous study knocking down the Imd pathway transcription factor Rel2 depicted a reduction in survival, indicating a protective effect of Imd pathway activation (Ramirez et al., B. bassiana at the level of PGRP-LC rather than a general mosquito antifungal response. The degree and specificity of this potential interaction between B. bassiana and PGRP-LC remains to be elucidated.The slight but significant increase in survival of I. javanica than those infected with B. bassiana. This is in agreement with our survival phenotypes and reiterates that while fungal detection by PGRP, and its subsequent downstream signaling through the immune pathways, has a stronger protective effect on mosquitoes infected with I. javanica, it has a very limited effect on infection with B. bassiana. This likely suggests a stronger ability of B. bassiana to circumvent the mosquito antifungal immune response. Whether this is due to active immune-suppressive activity via secondary metabolites or due to the inherent resistance of B. bassiana to the immune-derived effectors produced via PGRP elicitation, remains to be elucidated. Furthermore, our study only evaluated two fungal entomopathogenic species and it is quite possible that different fungal strains of B. bassiana will present different infection phenotypes. Thus, further work evaluating the role of PGRPs in infections with different strains is needed to confirm whether this phenotypic response is solely a characteristic of B. bassiana.Further assays evaluating the fungal load in PGRP-depleted mosquitoes confirmed the participation of PGRPs in the antifungal host response, whose dynamics are in turn dependent on the strain of invading fungal entomopathogen. For instance, our study showed a much greater and significant increase in fungal load in mosquitoes infected with In summary, our study reveals that in the complex interaction that exists between the mosquito immune system and the invading entomopathogenic fungi, PGRPs play a critical role in antifungal defense. Our data further indicates that the effectiveness of PGPR-based fungal detection/response varies according to the infecting fungal species and displays a temporal elicitation of PGRP genes during the early stages of infection. Information derived from our understanding of how mosquitoes detect and respond to microbes, and the mechanisms underlying host resistance to fungal entomopathogens, can improve mosquito control strategies using fungal entomopathogens by facilitating the selection of strains with different modes of action.All datasets generated for this study are included in the article/JR conceived and conducted the study, analyzed the data, and wrote the manuscript. EM assisted on the determination, analysis of time-dependent mortality data, and manuscript writing. LF-W and KV assisted with electron microscopy imaging and manuscript writing. AR assisted with data analysis and interpretation. All authors edited and approved the final 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.


Symptom remission at endpoint  was most closely aligned with a ≥46% reduction in ADHD-RS-IV total score. Clinical improvement was most closely aligned with a ≥40% reduction in ADHD-RS-IV total score. The three different measures of outcome were strongly aligned during double blind and open label treatment, and were independent of subtype status. Our data suggest that at least 40% improvement in symptoms is needed to achieve a robust response at endpoint. Clinical trials in attention-deficit/hyperactivity disorder (ADHD) have typically measured outcome using clinician ratings on the Attention-Deficit/Hyperactivity Disorder Rating Scale, Fourth Edition (ADHD-RS-IV) and the Clinical Global Impression-Improvement (CGI-I) scale. Remission has been defined as an endpoint score of less than or equal to 18 on the ADHD-RS-IV (or a mean score of 1). Responders have been defined as patients who achieve a CGI-I score of much or very much improved (1 or 2). There is a lack of agreement in the literature on what percent change in symptoms on the ADHD-RS-IV should be used to define improvement or remission. This study uses data from a clinical trial of a methylphenidate extended release (MPH-MLR; Aptensio XR Clinical trials of medication and psychological treatments for attention-deficit/hyperactivity disorder (ADHD) have looked at outcome in terms of symptom change from baseline, overall clinical improvement, and final symptom endpoint. Each of these provides a slightly different perspective on treatment response. For example, percent decrease in symptom scores reflects change from baseline. Clinical global impression of improvement also reflects change from baseline, but goes beyond core symptoms. Remission reflects patient achievement of a final outcome that would be considered “normalization”. Endpoint scores are distinct from change scores in that if someone starts treatment very ill  and has a 50% drop in symptoms, they may still be moderately symptomatic. Past trials have used arbitrary cutoffs for percent change in symptoms ranging from 25% to 40%. The importance of differentiating response in terms of improvement vs. remission first became apparent in a post hoc reanalysis of the Multimodal Treatment Study of Children with ADHD study . DiffereThroughout the various versions of the Diagnostic and Statistical Manual of Mental Disorders (DSM), rating scales for ADHD have scored the 18 DSM symptoms of ADHD on a 4-point Likert scale. The anchor points for measurement of symptoms have been either based on frequency  rates each of the 18 DSM, Fourth Edition (DSM-IV) ADHD symptoms as never or rarely = 0, sometimes = 1, often = 2, and very often = 3 . The DSMClinicalTrials.gov identifier NCT01239030) were utilized to investigate various measures of clinical response and symptomatic remission associated with MPH-MLR treatment in children and adolescents with ADHD.A wide range of definitions and assessment tools have been used to try to define treatment response and disease remission; medication trials in children have used a variety of endpoints to describe these terms ,9,10,11.MLR-MPH is a once-daily beaded formulation composed of an immediate-release layer and a delayed-release layer. Administration results in a rapid rise in plasma levels after oral administration with an onset of action within 1 h and a duration of action of 12 h. MLR-MPH has a bimodal release such that the outer layer is equal to that of immediate-release MPH with initial rapid dissolution and absorption of 40% of the dose, followed by an evenly maintained release of MPH through the day from the second layer.The relationship between percent change in the ADHD-RS-IV total score versus a predefined symptom threshold cutoff of ADHD-RS-IV total score ≤18 (symptomatic remission) versus a Clinical Global Impression (CGI)-Improvement (CGI-I) score of much or very much improved was explored to further understand the relationship and agreement between these outcomes. Furthermore, whether these relationships are similar for the combined versus inattentive ADHD subtypes will be explored. There were not enough patients with hyperactive/impulsive subtype to power inclusion of this group.The design and primary results of this phase III, randomized, double-blind, placebo-controlled, parallel, fixed-dose study of MPH-MLR safety and efficacy have been previously published . BrieflyThis study was conducted in compliance with the Good Clinical Practice guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, the US Code of Federal Regulations that relate to clinical trial conduct, and the principles of the Declaration of Helsinki. An institutional review board for each of the 16 study sites reviewed and approved the protocol, amendments, and informed consent.ADHD symptoms were measured using the ADHD-RS-IV . The ADHThe CGI is a measure that is used as a standard in clinical trials to reflect the clinician’s initial global impression of patient severity (CGI-S) at baseline and improvement with treatment (CGI-I). The CGI-S  is a 7-pRemission has been variously defined as a mean score of ≤1 of all 18 items  on the AThis post hoc analysis determined the percentage reduction in ADHD-RS-IV total score that best aligned with CGI-I score ≤2 (very much or much improved). For each visit after baseline, the percentage reduction in ADHD-RS-IV total score was calculated as:The percentage reduction that best aligned with CGI-I score ≤2 was determined as follows. For each visit, for each percentage reduction (ranging from 30–50%), the percentage of patients with at least the given reduction was compared with the percentage of patients with CGI-I score ≤2, and the percentage of patients with agreement between the 2 measures was calculated. The range was derived from the consensus that 30% has been described as the lower limit defining improvement, and 50% as the upper limit defining remission. For example, perfect agreement for a reduction of ≥30% and CGI-I score ≤2 would occur if all patients with a reduction of ≥30% had CGI-I score ≤2 and all patients with a reduction of <30% had CGI-I score >2. The 95% confidence interval (CI) for the percentage of patients with agreement was calculated, and McNemar’s exact test was used to compare the percentage of patients with at least the given reduction to the percentage of patients with CGI-I score ≤2. The percentage reduction that best aligned was the one with the greatest percentage of patients with agreement. Fisher’s exact test was used to compare the combined and inattentive subtypes using the percentage of patients with agreement of combined and inattentive ADHD subtypes.This post hoc analysis also determined the percentage reduction in ADHD-RS-IV total score that best aligned with ADHD-RS-IV total score ≤18 with a similar method.All calculations utilized SAS 9.4 .At least 1 dose of the study drug was taken by 230 enrolled patients (safety population), 221 patients were included in the evaluable population, and 200 patients completed the open-label dose-optimization phase. Two-thirds of the sample were stimulant naïve, and one-third of the sample (75/221) had a medication washout. The option to enter the open-label compassionate use extension was exercised by 173 patients, with 128, 112, 100, and 66 still in the study after visits 15, 16, 17, and 18, respectively. The open-label compassionate use extension encompassed up to 21 months of treatment. Combined was the most common ADHD subtype .After 7 days of double-blind fixed-dose treatment with no initial dose titration, 45% of the 221 patients achieved ≥30% symptomatic improvement, 34% achieved ≥40% symptomatic improvement, 24% achieved ≥50% symptomatic improvement, 30% had clinical response as defined by CGI-I score ≤2, and 29% were in remission with an ADHD-RS-IV total score ≤18. In general, the degree of clinical response and symptomatic remission achieved at the end of the 1-week double-blind fixed-dose phase was highly dose related, with symptomatic remission ranging from 21% at a dose of 10 mg daily to 40% at a dose of 40 mg daily . p = 0.26). Agreement between these measures was evident in 87% of patients , 82–91%). Percentage of patients with agreement was similar for combined (117/134 [87%]) and inattentive (63/72 [88%]) subtypes . At double-blind fixed-dose treatment end, a CGI-I score ≤2 was best aligned with a 40% reduction in ADHD-RS-IV total score, with CGI-I score ≤2 in 30% of patients and a ≥40% reduction in ADHD-RS-IV total score in 34% of patients, and the percentage of patients was not statistically different for these 2 measures . There was agreement between these measures in 88% of patients . The percentage of patients with agreement was similar for combined (120/134 [90%]) and inattentive (61/72 [85%]) subtypes .At the end of double-blind fixed dose treatment, remission  most closely aligned with a 46% reduction in ADHD-RS-IV total score, with remission in 29% of patients and a 46% reduction in ADHD-RS-IV total score in 27% of patients, and the percentage of patients was not statistically different for these 2 measures .At the end of the 11-week open-label dose-optimization phase, most patients had responded to treatment; 94% (188/200), 85% (170/200), and 73% (146/200) of patients receiving MPH-MLR had a ≥30%, ≥40%, and ≥50% reduction in ADHD-RS-IV total score, respectively. Clinical response, defined as a CGI-I score ≤2, was achieved in 92% (183/200) of patients. Symptomatic remission  was achieved in 75% of patients. Throughout the open-label dose-optimization phase, CGI-I score ≤2 remained closely aligned, with a 40% reduction in ADHD-RS-IV total score . At the Throughout the open-label compassionate use extension, overall response and remission rates were maintained by the majority of patients. There was sustained alignment between symptomatic response  and the clinician’s global assessment of being much or very much improved, with CGI-I scores ≤2 . SymptomThroughout various phases of the study, the percentage of patients achieving 40% symptom improvement was in best agreement with achieving CGI-I response. A 46% symptom improvement was in best agreement with symptom remission. While ~90% of patients in this trial were able to achieve some symptom improvement , it should be noted that only ~75% were able to achieve symptom remission. This pattern of response, in which most patients achieve improvement and a majority achieve remission, is consistent with other stimulant trials ,16,17. These data help to clarify our understanding of the relationship between symptom change, clinical improvement and symptom remission. Although each of these outcomes describes a slightly different way of looking at response, this study demonstrates that these outcomes are closely aligned. Our results emphasize the importance of targeting remission rather than simply response as the standard of care. Most important, this study provides empirical criteria to define the percent of change that should be considered as the convention for response in clinical trials. Previous definitions of symptom response that used 30% change as an acceptable measure of outcome probably were generous in identifying whether patients showed some degree of palliation with intervention, but may have contributed to clinicians failing to treat to optimization. Further research needs to look at how these definitions of symptom response correlate with actual functional improvement and remission. Importantly, analyses of data from this pivotal trial demonstrated that the agreement of CGI-I score ≤2 and a 40% reduction in ADHD-RS-IV total score held for patients with combined and predominantly inattentive subtypes. The agreement between and ADHD-RS-IV total score ≤18 and a ≥46% reduction in ADHD-RS-IV total score also held for both of these subtypes. This likely reflects the fact that the majority of patients in the trial had challenges in both domains. Clinicians should be cautious in looking only at total scores for 18 items, when looking at outcome in individuals where problems are largely limited to either attention or hyperactive/impulsive difficulty. For example, if a patient has a mean score of 2 for 9 symptoms of just hyperactive or just inattentive symptoms, they would be clinically moderately ill, but would still meet the criteria for “remission” defined as an endpoint score of 18.A robust standard for fine tuning a high standard of treatment response, as per the methodology used by Swanson in comparing treatments in the Multimodal Treatment of ADHD (MTA) study , would dThis study has several limitations. This is an exploratory post hoc analysis using data from a pivotal trial that included a representative ADHD patient population and used a phase III, double-blind, parallel, fixed-dose, randomized design, followed by open label. Given the conventional methodology of the trial, this is a reasonable sample with which to examine the relationship between outcome and measures. Nonetheless, the results are representative of the particulars of this design and patient population and need to be replicated in other types of trial design, populations, and types of treatment intervention. Two-thirds of the sample were stimulant naïve, and it is unclear how this may have impacted patient perception of symptoms. Although we had a full range of responses, partly because the double-blind period was short and dose was randomized rather than optimized, the high agreement between outcomes we obtained in open-label follow-up may reflect a limitation of the high rate of response in open label follow ups, given that non-responders would not typically stay in the study. It should also be noted that all of these measures are completed by clinicians and have a certain degree of overlap. For example, the clinician is aware of the ADHD-RS rating when completing the CGI, so our results do not reflect truly independent measures or interrater reliability. No special training was done to standardize clinician ratings, but again this is not atypical of clinical trials conduct. Nonetheless, this is how these outcomes are completed in clinical trials in ADHD and reflect real world practice. Further research on standardizing appropriate clinical trial outcomes in ADHD, avoiding redundancy between scale outcomes that overlap, and empirical definition of response vs. remission are needed. This research would require receiver operating characteristics to determine the most sensitive cut points, and logistic regression analysis to compare across measures and outcomes. Other limitations of this study reflect limitations of current methodology for measuring outcome in ADHD clinical trials in general. First, although by definition remission should be considered as per the work of Biederman et al.  as both This is the first study we are aware of that examines the relationship between different definitions of symptom improvement and symptom remission and specifies empirically the percent change in symptoms associated with each of these outcome targets. High rates of clinical response to stimulant treatment in patients with ADHD have been well documented. This study identifies an empirical definition of percent symptom change that can be used as a standard to identify success in achieving optimal outcomes, and a way of differentiating small differences across trials that otherwise might not be apparent.


Tunisia, being part of the secondary center of diversity for durum wheat, has rich unexploited landraces that are being continuously lost and replaced by high yielding modern cultivars. This study aimed to investigate the genetic diversity and population structure of 196 durum wheat lines issued from landraces collected from Tunisia using Diversity Array Technology sequencing (DArTseq) and to understand possible ways of introduction in comparing them to landraces from surrounding countries. A total of 16,148 polymorphic DArTseq markers covering equally the A and B genomes were effective to assess the genetic diversity and to classify the accessions. Cluster analysis and discriminant analysis of principal components (DAPC) allowed us to distinguish five distinct groups that matched well with the farmer’s variety nomenclature. Interestingly, Mahmoudi and Biskri landraces constitute the same gene pool while Jenah Zarzoura constitutes a completely different group. Analysis of molecular variance (AMOVA) showed that the genetic variation was among rather than within the landraces. DAPC analysis of the Tunisian, Mediterranean and West Asian landraces confirmed our previous population structure and showed a genetic similarity between the Tunisian and the North African landraces with the exception of Jenah Zarzoura being the most distant. The genomic characterization of the Tunisian collection will enhance their conservation and sustainable use. Durum wheat is the tenth most important crop worldwide, grown on 13 M ha with a production of 39.1 Mt in 2017  and is mainly used for pasta production. It is mainly grown in the countries around the Mediterranean basin and its cultivation has extended to India, Mexico, North America, and Russia .Triticum turgidum ssp. durum) is a self-pollinated allotetraploid cereal  originating from a cross between Aegilops speltoides (SS) and Triticum urartu (AA) and domesticated from primitive wheat (Triticum turgidum ssp. dicoccum) in the Fertile Crescent around 8000 years ago [Durum wheat  contains 60,488 durum wheat accessions of which 22,600 are landraces. The International Center for Agricultural Research in the Dry Areas (ICARDA) genebank holds one of the largest collections of durum wheat accounting more than 20,531 accessions of which more than 65% are landraces. The Tunisian genebank (NGBT) holds a total of 4000 accessions of durum wheat most of which are pure lines collected from the predominant remaining landraces.Durum wheat landraces are valuable parental germplasm for wheat improvement programs around the world. Genesys database  characterizing for the first time the population structure and the genetic diversity within and among the Tunisian durum wheat collection, (2) looking at potential mis-classification by linking off types of landraces to local modern durum wheat varieties and/or other landraces, and (3) comparing the Tunisian durum wheat landraces to landraces from the countries around the Mediterranean basin and West Asia region using high throughput DArTseq™ technology.In this study, a total of 110,856 DArTseq markers were identified in a set of 196 lines issued from six Tunisian durum wheat landraces, of which 16,148 markers were found to be of high quality and polymorphic, after removing markers with more than 20% missing data and with less than 5% minor allele frequency (MAF). Around 10% of the markers had 0 heterozygote alleles. The average of the polymorphism information content (PIC) value of the DArTseq markers was 0.165, and the median was 0.105. The distribution of the majority of MAFs was between 0.05 and 0.15 .https://urgi.versailles.inra.fr/download/iwgsc/). The distribution is almost equal between A (4201 markers) and B (4659 markers) genomes with the highest number of markers observed on the chromosomes 7A and 2B. However, 7324 markers are still unassigned to any of the chromosomes (He) is observed for both A and B genomes with an average of around 0.25. The maximum value of the expected heterozygosity (He) was observed at chromosome 6B (0.28) and the minimum value was seen for chromosomes 3A, 4A, 5A, and 5B (0.24). For all chromosomes, the expected heterozygosity values (He) were higher than the observed heterozygosity values (H0).The DArTseq markers are well distributed across all the 14 chromosomes of durum wheat genomes based on the consensus bread wheat genetic map obtained from the International Wheat Genome Sequencing Consortium database  among the 196 Tunisian landraces lines ranged from 0 to 0.79 with an average of 0.46 . HoweverCluster analysis of the 196 lines derived from the six Tunisian landraces was performed using the allele sharing distance (ASD) method and the results allowed us to group them into five clusters and to identify lines wrongly assigned to some landraces. The first cluster comprised all the lines of Jenah Zarzoura (Zar). The second cluster contained mainly Bidi lines (Bid). The third cluster is subdivided into two sub-groups, one containing Rommani lines (Rom) and the other having a mixture of lines from other landraces (seven from Biskri (Bis), three from Jenah Khotifa (jkf), and two from Bidi (Bid)). Cluster 4 had the majority of Jenah Khotifa lines (jkf) but contained also four lines of Rommani, two of Biskri, and one of Mahmoudi. The last cluster constitutes the largest one and gathered almost all lines of Biskri (Bis) and Mahmoudi (Mah) .The DAPC  results showed that 88 Principal Components (PCs) explain 82% of the total molecular variance. The optimum number of clusters was obtained with K = 6 using the Bayesian Information Criterion (BIC), which divided the lines into six sub-populations . The firIncreasing K to 6 resulted into the formation of an additional small group composed by 13 lines mainly of Biskri (7) and Jenah Khotifa (4) showing that these lines could be off-types within their respective landraces, which need further characterization E. The glThe results of analysis of molecular variance (AMOVA) for K = 2 to K = 7 showed that the variance between populations was high for K = 2 (31.92%), and it decreased for K = 3 and K = 4  to increase and reach a plateau for K = 5 with a slightly higher value (31.77%). The variance between lines within populations decreased from 11.32% for K = 2 to reach values close to 0% for K = 5. Comparing AMOVA results between populations with K = 5 and the real groups using the landrace’s name clearly support the hypothesis that K = 5 better differentiates genetically the lines than the characterization of landraces by name, resulting in higher variance between populations , lower variance between lines 68.23% for K = 5 and 70.39% for real groups, and lower variance between lines within populations . Thus, tResults from hierarchical AMOVA using hierarchical subdivision strata from K = 2 up to K = 5 indicated that most of the molecular variance was explained with the first level K = 2 separating Jenah Zarzoura from the other landraces (17.18%) and the fifth level K = 5 within the four groups (26.62%) separating Bidi, Rommani, and Jenah Khotifa and the large group constituted by Biskri and Mahmoudi. The remaining molecular variance (variance between lines) was also low compared to non-hierarchical AMOVA results (60.55%) using K = 5 .Finally, the resulted variance components from AMOVA analysis with K = 5 were significant when they were tested using permutations . These rThe genetic distance between landrace populations showed that Jenah Zarzoura sub-population is the farthest from all the other landraces, from Rommani (0.820), from Biskri and Mahmoudi (0.730), from Jenah Khotifa (0.682), and from Bidi (0.816) . FurtherHe = 0.27). The lowest genetic diversity was observed within the Bidi population (He = 0.12). Biskri and Mahmoudi forming the same group with 73 lines showed a moderate genetic diversity (He = 0.23). Based on FST values, Jenah Zarzoura showed a low FST value of 0.05. Thus, this population presents a genetic drift compared to the others, having a fixation of alternate alleles with FST values superior to 0.25 , Jenah Khotifa (jkf), and Bidi (Bid) were grouped with the majority of the Algerian landraces (8); Sub-population 12 included all Ethiopian landraces (11), along with one accession from Afghanistan and two accessions from Yemen; Sub-population 9 included the Tunisian landraces Biskri (Bis) and Mahmoudi (Mah) that were grouped with the remaining five landraces from Tunisia, with the majority of Algerian and Lebanese landraces (6), and with some Moroccan landraces (3). Sub-population 11 included exclusively the Tunisian landrace Jenah Zarzoura (Zar) with two accessions from Jordan . The popThe molecular markers techniques are important tools for better understanding genetic diversity, undertaking association mapping and ensuring efficient conservation and management of genetic resources. This study demonstrated the relevance of DArTseq technology as a reliable and cost-effective tool for assessing the diversity within and between landraces and for comparing them with other germplasm of durum wheat. This technique yielded a large number of polymorphic and informative markers equally spread in the A and B genomes allowing high coverage of the genomes of the Tunisian durum wheat germplasm compared to other molecular techniques used previously. Similar genomes coverage was found on a panel of 170 durum wheat entries . The larPolymorphic information content (PIC) values revealed by DArTseq markers averaged 0.165 with an asymmetric distribution skewed towards low values. The same distribution tendency was found using the same approach for 91 durum wheat landraces from the Fertile Crescent and for 138 wheat germplasm from Southwest China ,41. Ren He = 0.25), which is higher than the one exhibited by a set of world-wide durum wheat collection (He = 0.224) using SNP markers [The lines of the six Tunisian durum wheat landraces showed a moderate level of genetic diversity  and DAPC  were used for depicting the genetic relationship and structure of the Tunisian collection. The first method classified the panel into five groups matching mostly with the farmer’s landraces names, with Jenah Zarzoura being the most distant and Mahmoudi and Biskri included in the same cluster. The closeness of Mahmoudi and Biskri is for a long time reported by Boeuf  based onThe use of the multivariate method DAPC to evaluate the population structure showed better performance and allowed for a population subdivision similar to other studies ,48. SeveHierarchical AMOVA analysis based on hierarchical subdivision strata from K = 2 up to K = 5 agreed with DAPC analysis results and supported the high level of molecular variance to K = 2 (17.18%) and to K = 5 (26.62%). AMOVA analysis results on the basis of the landraces’ names indicated a higher genetic variation among (28.79%) rather than within (0.82%) Tunisian landraces. Taking in consideration the structure of the population based on the optimal number of grouping K = 5, AMOVA results showed an increase in percent of the explained genetic variance among landraces (~31.77%) and a decrease of the genetic variance within them. Soriano et al.  revealedHe = 0.27), and the Bidi landrace showed the lowest genetic diversity (He = 0.12). The low genetic variation within the landraces could be explained by the selection from farmers for desirable traits and/or from environmental conditions pressure. Compared to the other landraces, the Jenah Zarzoura landrace showed a high expected heterozygosity (He = 0.268) and a low fixation index (FST = 0.05). Thus, this small differentiation could be explained by the confinement of this landrace to a specific environment in the oasis of Mareth, which reflects the geographic isolation of the oasis of Mareth, limited seed exchange, and selection pressure by farmers. Most often, farmers are selecting the best representative spikes from a landrace to form the seed lots.This study allowed us to assess for the first time the genetic diversity within and among the Tunisian durum wheat landraces using the DArTseq technique, which allowed us to show different levels of genetic diversity within landraces. Jenah Zarzoura and Rommani populations showed the highest genetic diversity . These results confirm the possibility of mixture in some landraces, which could be due to seed exchange and threshing practices as suggested by other studies ,54. ThisTunisia is considered as a secondary center of diversity for durum wheat, and the introduction patterns of durum wheat landraces into Tunisia and North Africa are still under discussion. The DAPC analysis including landraces from Tunisia and landraces from the countries around the Mediterranean and West Asia regions allowed us to define 12 distinct groups, which can be used to highlight the relationships between Tunisian landraces and other landraces. Most Tunisian landraces held at the ICARDA genebank as well as the lines derived from landraces collected recently in Tunisia were grouped with landraces from North Africa neighboring countries and with landraces from Greece, Italy, and Lebanon. Jenah Zarzoura remained distinct from all landraces studied except for the two landraces from Jordan. These results suggest that most Tunisian landraces could be obtained through Lebanon via Greece and Italy, while Jenah Zarzoura was obtained through another introduction pathway. Previous reports have demonstrated two dispersal patterns of durum wheat in the Mediterranean Basin from the Fertile Crescent: over the north side via Turkey, Greece, and Italy and the south side via North Africa . MoragueThe results of this study can be used to direct future activities of collection, conservation, and the use of durum wheat genetic resources in Tunisia. In terms of adding new diversity to the existing collections, more collection is needed in Tunisia mainly in the oasis areas to collect different landraces like Jenah Zarzoura. Future studies of this kind of germplasm will shed more light on the specific nature of this germplasm as in the case of durum wheat germplasm from Ethiopia . AdditioSet 1: A total of 196 pure lines issued from six Tunisian durum wheat landraces known as Mahmoudi, Rommani, Jenah Zarzoura, Bidi, Jenah Khotifa, and Biskri, collected from 5 regions between 2009 and 2010 and conserved by the National Genebank of Tunisia (NGBT), were used for assessing the intra and inter genetic diversity . The lanSet 2: A total of 40 accessions composed of six improved varieties released in Tunisia, seven ICARDA/CIMMYT elite inbred lines and 27 Tunisian durum wheat landraces  were used for identification of potential mis-classified lines from Set 1 and for comparison between Tunisian durum wheat landraces and improved germplasm .Set 3: A total of 207 durum wheat landraces collected from Mediterranean and West Asia countries—Morocco (17), Algeria (14), Tunisia (13), Libya (9), Egypt (12), Lebanon (10), Syria (11), Jordan (11), Israel/Palestine (10), Iraq (11), Iran (12), Afghanistan (16), Yemen (10), Greece (18), Cyprus (10), Italy (12), and Ethiopia (11)—randomly chosen from the ICARDA genebank collection along with four lines representing each Tunisian landraces from Set 1 were used for a genetic relationship study  .This study used three sets of germplasm:The seeds for Sets 1 and 2 were taken from single plant spikes of each line, landrace, and germplasm grown at the Mornag INRA-Tunisia experiment station during the 2014–2015 growing season, while the seeds for Set 3 were send to CIMMYT from the ICARDA genebank within the joint effort to genotype wheat genetic resources.Fresh young leaves from a single individual plant per accession have been used for genomic DNA extraction performed through a modified CTAB (cetyltrimethylammonium bromide) method . The DNATM technology was employed to generate a genomic profile of the germplasm at the Genetic Analysis Service for Agriculture (SAGA) facility at CIMMYT, Mexico. This method used a combination of two restriction enzymes (PstI and HpaII) to reduce the genome complexity and to generate a genomic representation of the samples [A high-throughput genotyping method using DArTseq samples . The genwww.diversityarrays.com) developed by DArT Pty. Ltd., Australia, and the reference wheat genome issued from the International Wheat Genome Sequencing Consortium database (IWGSC WGAv0.4), available online at https://urgi.versailles.inra.fr/download/iwgsc/.DArTseq markers were mapped using the consensus map version 4.0  [The summary statistics of the filtered DArTseq markers such as the expected heterozygosity (He) or genetic diversity (GD), minor allele frequency (MAF), and the polymorphic information content (PIC) , were cact.org/) .For cluster analysis of the collection, allele sharing distance matrix was computed as described by Goa et al. . The dislgorithm . The clulgorithm .adegenet package and popr in R-project [K-value with the lowest BIC was retained as the optimal number of clusters. A discriminant analysis was then implemented using the groups found by k-mean clustering [Discriminant analysis of principal components (DAPC) was used to infer the number of clusters of genetically related individuals , using t-project . DAPC isustering .randtest function in the R-project as described by Excoffier et al. [For detecting the genetic variation among and within population(s) and supporting the hypothesis of the population structure, analysis of molecular variance (AMOVA) was performed for different hierarchical subdivision levels as well as for the full population structure strata from K = 2 to K = 8. Significance levels for variance component were estimated based on 10,000 permutations using the r et al. .Ho, the proportion of loci that are heterozygote for a population), the expected heterozygosity or genetic diversity , and the F-statistics (FST) as developed by Wright [For genetic differentiation and relationships among the sub-populations issued from the population structure analysis, the genetic distance between the sub-populations using Reynolds genetic distance was computed , and they Wright , were ca