To determine whether the presence of vitreomacular interface abnormalities (VMIA) in patients with diabetic macular oedema (DMO) modifies the response to ranibizumab.Medical records and spectral-domain optical coherence tomography (SD-OCT) scans of consecutive patients with centre-involving DMO initiating therapy with ranibizumab between December 2013 and March 2014 at the Belfast Health and Social Care Trust were reviewed. Patients were identified through an electronic database. Demographics; systemic baseline characteristics; history of previous ocular surgery/laser; best-corrected visual acuity (BCVA), central retinal thickness (CRT) and stage of retinopathy at presentation; and BCVA, CRT and presence/absence of fluid at the last follow-up were recorded. OCT scans were reviewed by a masked investigator who graded them for the presence/absence of VMIA at baseline and during follow-up and for the change in the posterior hyaloid face during follow-up. The association between (1) VMIA at baseline and (2) the change in the posterior hyaloid face during the follow-up and functional/anatomical outcomes was evaluated.p = 0.007), previous macular laser and panretinal photocoagulation (PRP) (p = 0.006) and previous cataract surgery (p = 0.01) between eyes with and without VMIA, with better levels of vision, higher frequency of macular laser and lower frequency of PRP in eyes where no VMIA was present. Multivariable regression analysis did not disclose any statistically significant associations between VMIA at baseline or change in the posterior hyaloid face during the follow-up and functional and anatomical outcomes following treatment.One hundred forty-six eyes of 100 patients (mean age 63.5 years) followed for a mean of 9 months  were included. Statistically significant differences were observed at baseline in BCVA (VMIA are associated with worse presenting vision in patients with DMO; VMIA or change in the posterior hyaloid face during the follow-up did not modify the response to ranibizumab in this study. The vitreomacular interface (VMI) gained greater scientific interest since the advent of optical coherence tomography (OCT). Spectral-domain OCT (SD-OCT) allows excellent visualisation of the VMI, enabling the study of the role of VMI abnormalities (VMIA) in the development of macular disease and the response to treatment. Vitreomacular traction (VMT), a form of VMIA , has beeThe reported prevalence of VMIA in patients with DMO ranges from 6.6 to 52.1%, depending on the diagnostic criteria and retinal imaging modality used . Only a Anti-vascular endothelial growth factor (anti-VEGF) agents have revolutionised the management of DMO. Clinical studies exploring the effect of the presence or absence of VMIA on the response to anti-VEGF treatment in DMO patients are scarce , 11. EluIntravitreal injections of anti-VEGFs (or other substances) may alter the anatomical structure of the vitreous leading to changes in the status of the posterior hyaloid , 12. TheHerein, we present findings on a large cohort of patients receiving intravitreal anti-VEGF therapy with ranibizumab for DMO and investigate the potential relationship between (1) VMIA at baseline and (2) the change in the posterior hyaloid face during the follow-up and functional/anatomical outcomes following anti-VEGF therapy.The medical records of consecutive patients with centre-involving DMO that met the eligibility criteria for intravitreal ranibizumab treatment as per the UK National Institute for Health and Clinical Excellence (NICE) guidelines  and who In keeping with the standard clinical practice at our site, all patients initiating anti-VEGF therapy received an ocular examination, which included refraction undertaken by an optometrist and best-corrected visual acuity (BCVA) measured with Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity charts at baseline, visual acuity testing obtained using ETDRS visual acuity charts and, following the refraction obtained at baseline, intraocular pressure measurement and slit-lamp biomicroscopy. SD-OCT scans were routinely obtained by trained ophthalmic photographers at every clinic visit using commercially available equipment . SD-OCTs were obtained in the standard mode .Grading of the diabetic retinopathy was determined by the ophthalmologists evaluating the patients in the clinic. Macular oedema was classified, based on OCT findings, into mild, moderate and severe as described by the Global Diabetic Retinopathy Project Group . GradingAll intravitreal anti-VEGF injections were performed in a clean room with topical povidone 5% and topical anaesthetic before the procedure. The treatment protocol involved three-monthly ranibizumab injections followed by a pro re nata regimen thereafter.SD-OCT scans were used to obtain data on central retinal thickness (CRT) values. The CRT was computed automatically using built-in retinal mapping software. SD-OCT video clips  from all included patients were evaluated by a senior investigator (NL) masked to clinical findings, including visual acuity and treatments received; the status of the posterior hyaloid and VMI at baseline and at the last follow-up was graded (see below) and recorded.SD-OCT video clips enabled detection of foveal and extra-foveal traction as well as changes outside the central macular area and also differentiation between epiretinal membranes and taut posterior hyaloid. The SD-OCT appearance of the inner retinal surface was graded based on the International Vitreomacular Traction Study Group classification of vitreomacular adhesion, traction, and macular hole as follows: (a) normal VMA, (b) VMT Fig. a, (c) fuThe status of the posterior hyaloid face (PHF) was also graded based on the presence or absence of a PHF attachment as (a) totally attached Fig. a, (b) pat test.The statistical analysis of the data was performed using the Statistical Package for the Social Sciences, Windows version 21 . The distribution of continuous variables was assessed for normality using the Kolmogorov-Smirnov test, and the transformation was performed, if necessary, to achieve normal distribution. Categorical variables were compared using Pearson’s chi-square test. Continuous variables were compared using the independent samples p values < 0.05 were considered statistically significant.General estimating equations (GEE) were used to undertake the multivariable regression analysis to enable data from both eyes to be included. GEE models account for the correlation between two eyes from one patient. A multivariable modelling controlled for age and gender was undertaken. A second model was also run controlling for those covariates shown to be significant in the univariate analysis. In patients with both eyes meeting eligibility criteria, data from both eyes were used. The authors confirm that data collection conformed to the local policy at the Belfast Health and Social Care Trust; this study was registered with the audit department (number 4966).The study cohort included 146 eyes of 100 consecutive patients with a mean age of 63.5 years at the time of presentation (range 24–88 years). One hundred thirty-eight eyes (94.5%) exhibited severe macular oedema, which includes retinal thickening or hard exudates involving the centre of the fovea. Eighty-two eyes (56.1%) had no previous macular laser treatment at baseline. The descriptive characteristics of the 100 patients (146 eyes) studied are shown in Table p = 0.007). Eyes with VMIA were more likely to have undergone panretinal photocoagulation but less likely to have had macular laser than eyes without VMIA (p = 0.006). There was no association between CRT at presentation and the presence of VMIA. A higher proportion of patients with VMIA had undergone cataract surgery (p = 0.01). There was no association between the presence of VMIA and the duration of diabetes, HbA1C level, history of hypertension or kidney disease.Signs of VMIA were detected in 28 eyes (18.5%); 19 of these (70%) had an ERM, 8 (28.5%) had VMT and one (0.5%) had both VMT and ERM. A statistically significant difference in mean BCVA was observed between patients with and without VMIA, with patients with VMIA having worse vision  was the only factor found to be statistically significantly associated with this occurrence.Partial or complete detachment of the posterior hyaloid interface was observed in 22 eyes during the follow-up (15%). A history of kidney disease (p = 0.052) between posterior hyaloid detachment and absence of macular fluid at the last follow-up.Multivariable regression analysis did not reveal any statistically significant difference between detachment of the posterior hyaloid during follow-up and change in BCVA, number of injections per month, changes in CRT and status of macula at follow-up, when corrected for age, gender and history of kidney disease Table . There wIn the current study, VMIA, as defined by the International Vitreomacular Traction Study Group, were detected in 18.5% of patients with DMO, the majority of these being ERM (70%). VMIA were more common in patients that had previously undergone panretinal photocoagulation (PRP) and cataract surgery and less frequent in those that had received macular laser. Patients with VMIA presented with lower levels of vision. The presence of VMIA at baseline did not dampen the functional (VA) or structural (CRT) response to ranibizumab treatment.The prevalence of VMIA in patients with DMO identified in this study appears to be within the previously reported range Table  2, 6–10–106–10. Previous PRP was associated with the presence of VMIA. ERM formation may occur as a result of laser panretinal photocoagulation . It has VMIA were also identified more frequently in patients who had undergone cataract surgery. Pseudophakia is associated with an increased incidence of posterior vitreous detachment . FurtherLower visual acuity at presentation was found in patients with VMIA when compared with those without them. This agrees with findings of other studies , 11.Previous studies evaluating the effect of VMIA on the response to anti-VEGF agents have reported conflicting results. Further analysis of a Diabetic Retinopathy Clinical Research Network study found the absence of “surface wrinkling retinopathy”, as determined by colour fundus photography, to be associated with a significantly better visual outcome after one year of treatment with ranibizumab . HoweverA small study by Yoon et al. evaluated the effect of VMIA  on the response to anti-VEGF treatment in patients with DMO . Only teWong and colleagues recently reported that the presence of an ERM at presentation was predictive of a more limited functional  and anatomical (CRT) response in a study including 77 patients 104 eyes) with DMO [4 eyes win = 26) had a greater improvement in visual acuity than those without VMA (n = 98) at 6 months following treatment with ranibizumab [A retrospective cohort study of 124 eyes enrolled in the READ-3 trial found that eyes with evidence of vitreomacular adhesion at baseline  undergoing anti-VEGF therapy found the mean incidence of VMIA to be 6.43% per year . In thisPVD is more prevalent in eyes without DMO . There in = 6; Table This study has several limitations, including its retrospective nature, the relatively short follow-up period and the fact that cataract surgery during the study period was not systematically recorded. We believe, however, that it would be unlikely that patients included in this series would have received cataract surgery during the study period. The reasons for this are that it is a standard practice at our institution for patients with DMO to be treated first with anti-VEGFs and cataract surgery postponed until resolution of the DMO has occurred. Also it would be dffocult that paetients would hacve undergone cataract surgery suring the study period considering waiting times for cataract surgery and the length of the follow-up of the patients included in the study. Information about whether eyes received focal laser treatment or PRP during the period of the study was, similarly, not systematically recorded. It would be unlikely that patients would have received focal laser treatment during the study period as in our clinics, patients initiating anti-VEGF therapy would have continued on anti-VEGFs monthly, unless the fluid would have resolved, during the first year of treatment. A small proportion of eyes, however, would have received PRP for active PDR (The findings of our study would suggest that the presence of VMIA in patients with DMO does not appear to dampen the response to anti-VEGF therapy.


Differences in cognitive performance between males and females are well-described, most commonly in certain spatial and language tasks. Sex-related differences in cognition are relevant to the study of the neurotypical brain and to neuropsychiatric disorders, which exhibit prominent disparities in the incidence, prevalence and severity of symptoms between men and women. While structural dimorphism in the human brain is well-described, controversy exists regarding the existence and degree of sex-related differences in brain function. We analyzed resting-state functional MRI from 650 neurotypical young adults matched for age and sex to determine the degree of sexual dimorphism present in intrinsic functional networks. Multilevel modeling was pursued to create 8-, 24-, and 51-network models of whole-brain data to quantify sex-related effects in network activity with increasing resolution. We determined that sexual dimorphism is present in the majority of intrinsic brain networks and affects ∼0.5–2% of brain locations surveyed in the three whole-brain network models. It is particularly common in task-positive control networks and is pervasive among default mode networks. The size of sex-related effects varied by network but can be moderate or even large in size. Female > male effects were on average larger, but male > female effects spread across greater network territory. Using a novel methodology, we mapped dimorphic locations to meta-analytic association test maps derived from task fMRI, demonstrating that the neurocognitive footprint of intrinsic neural correlates is much larger in males. All results were replicated in a motion-matched sub-sample. Our findings argue that sex is an important biological variable in human brain function and suggest that observed differences in neurocognitive performance have identifiable intrinsic neural correlates. Research in human neuroscience and psychology has described many differences in the performance of neurocognitive tasks between males and females. More recently, a newer generation of studies has re-examined these findings in a more nuanced fashion, attempting to take into account the influence of potential co-varying factors such as gender identification and socialization. While this latter work has de-emphasized the wide scope of earlier findings, some types of cognitive tasks have been confirmed to show significant differences in performance between males and females. Broadly, males tend to exhibit superior performance in selected spatial and visuospatial tasks and are somewhat disproportionately represented among high performers in mathematics, whereas females perform better at reading and certain tasks of verbal fluency, recognition memory, and episodic memory . HoweverSex-related differences in cognition are of interest not only for the study of the neurotypical brain, but also in the context of neuropsychiatric disorders. Sex profoundly influences the prevalence, incidence and severity of various neuropsychiatric disorders. Well-known examples include the increased prevalence of attention deficit hyperactivity disorder (ADHD) and autism in males, vs. higher lifetime rates of depression and anxiety in females. The incidence of anxiety disorders accelerates faster in adolescent females, whereas schizophrenia onsets earlier in males, with a more severe course. Sex-related differences are also present in the diseases of senescence, with Alzheimer’s disproportionately affecting females, but Parkinson’s disease and Lewy body dementia being 2-4x more common in men . While aThe continuous development of non-invasive imaging technologies with good spatial resolution such as magnetic resonance imaging (MRI) has enabled the investigation of sex-related differences in human brain structure and function, building bridges between observed differences in cognitive performance and their potential neural correlates. Perhaps the most robust findings have been of a greater proportion of gray matter in females as compared to white matter and larger absolute brain volume in males, even after correcting for body size . The forin vivo using resting-state fMRI (rsfMRI). This activity is spatio-temporally organized, and replicable macroscale intrinsic neural networks have been identified with specific neurocognitive associations . In fMRI during task performance, sex-related differences have been observed paralleling the psychological literature, such as visuospatial tasks . In the ciations . Consideciations  or indivciations . Conversciations . Given tin silico mapping of statistical effects identified in intrinsic networks onto meta-analytic association test maps of neurocognitive functions. This approach allows the construction of a computational ‘bridge’ from brain networks observed during rsfMRI to neurocognitive maps derived from hundreds of task fMRI experiments.The relative ease of acquisition of rsfMRI render it a tractable medium for examining brain function across ages, species and cognitive levels. Further, macroscale intrinsic networks observed in the resting condition are similar to those detected during task performance , being tTo advance the debate on sex-related differences in intrinsic brain networks, we asked whether we could isolate male > female (M > F) and female > male (F > M) effects in intrinsic brain networks in a large sample of typically developing young adults matched for age and sex, and how these effects related to brain maps associated with nine neurocognitive functions with good evidence of sex-related performance differences. As noted above, sex-related performance differences have also historically been detected in many other cognitive tasks, albeit less consistently or less frequently reported. For comparison purposes, we therefore also elected to survey a selection of cognitive control functions, where there is a less robust evidence base for sex-related performance differences. Given the existing controversy in this field, we formulated a design where intrinsic brain networks were obtained using independent component analysis (ICA), a popular and very well-established  data-dri1. From this larger initiative, the originators formed and released a repository in 2015 comprising demographic, MRI and behavioral data from a subset of 1570 healthy young adults ages 18–35, where age was specified within 2-year bins. For example, the 19-year-old bin includes subjects aged 18 and 19 at the time of scanning. Our study uses data from subjects in this latter sample, where the “dispersion of estimated IQ scores [was] positively shifted relative to the general population” but personality traits “have distributions that would be expected of a clinically screen population-based sample  of functional MRI were acquired with 47 slices, interleaved sequence, voxel size 3.0 mm × 3.0 mm × 3.0 mm and TR = 3 s. Resting state scans were acquired with participants instructed to “remain still, stay awake and keep their eyes open while blinking normally” . A fixatnetworks . In addinetworks . Slice-bnetworks  and 88 pnetworks . All subp = 6.995 × 10-9), with males having higher average scores. Accordingly, we also created a sub-sample of 534 subjects that were similarly all right-handed and matched for age and sex but were also matched for head motion as defined by DVARS score. A list of the 136 subjects removed to create this motion-matched sample is in In this step, two samples of participants were prepared from the total of 1569 GSP subjects remaining after pre-processing (see MRI Pre-processing). The first was a 670-subject sample matched for age and sex, the second a 535-subject sample additionally matched for motion. The 670-subject sample for this study was constructed by selecting right-handed subjects with estimated IQ scores available, and then sex-matching within age bins. Subject demographics may be viewed in In this step, pre-processed rsfMRI data (see MRI Pre-processing) for the two subject groups (see Subject Sample Construction) was used to generate 8-, 24-, and 51 network models of brain function by submitting the pre-processed data to group spatial ICA . Gray matter components were identified in each of the 3 models by taking the entire output  of the group ICA and eliminating noise components . The resulting gray matter components were thresholded in order to construct spatial maps of each intrinsic network representing the strongest and most consistent coactivations between brain regions within a network . These spatial maps were used to attribute the neurocognitive labels for each IN , and served as the inputs for the remainder of the analyses. This process was followed for each brain map in each of the 3 ICA models.z-scores.Using the pre-processed rsfMRI data (see MRI Pre-processing), we performed group spatial ICA using the Group ICA of fMRI Toolbox (GIFT) developed in our group, and widely used in ICA of fMRI . ICA is Using the output of all components from each of the 3 ICA decompositions , we sorted components into gray-matter networks vs. artefactual noise components with a combination of expert visual inspection by NdL and VC, and quantitative metrics in order to isolate gray-matter or neural components. To do this we computed the quantitative spectral metrics of (1) Fractional amplitude of low frequency fluctuations and (2) Dynamic range  for evert-test on the individual subject timecourses and thresholding individual voxels at (mean + 4 standard deviations), again following an established pipeline (https://neurovault.org/collections/4030/ (8-network model), https://neurovault.org/collections/4031/ (24-network model) and https://neurovault.org/collections/4032/ (51-network model). Each intrinsic network is labeled with its attributed neurocognitive function and numbers, that correspond to After the sorting process  we constructed a spatial map for each gray-matter IN that had been retained to select voxels that represented the strongest and most consistent coactivations within each IN, by performing a voxelwise one-sample pipeline  using GI3. Thirdly, network correlations with association test maps of regional activations associated with specific neurocognitive functions were inspected in Neurosynth  associated with peak intensities for each IN in each of the 3 sets of maps. The top 3 co-ordinates were compared with the literature. We found multiple literature-based confirmatory sources that gave specific Talairach or MNI coordinates and associated these with network labels for all networks in the task-positive network group, the DMN and primary sensorimotor and visual networks  but not urosynth . AttribuIn this step, we used the group ICA output of 3 sets of labeled, thresholded spatial maps of INs (see Construction of Whole Brain Models of Intrinsic Networks) as the substrate for a multivariate statistical analysis aimed at finding sex-related differences in each IN in each of the 3 models of whole brain function. We first performed a multivariate analysis of covariance (MANCOVA) using the MANCOVAN toolbox and an established method  in GIFT,For each predictor that proved significant in the univariate analysis, the effect size (beta) was determined by computing connected voxel clusters (similarly to the bwlabeln function in MATLAB) and then calculating an average beta over the cluster of voxels. The fraction of the network map accounted for by each effect was determined by calculating the fraction of the total voxels in each network map represented by voxels with significant effects . The size and fraction of both F > M and M > F effects were computed for each of the 3 IN sets in both subject groups.In this final step, we used the output of the multivariate analysis  which identified significant sex-related effects in each IN in each model as the input to a mapping process where we mapped effects of sex in each IN to neurocognitive functional maps using a method which we recently developed and published . The aim4 uses text mining to identify terms of interest  within neuroscience articles occurring at a frequency of > 1/10,000 words, and extracts fMRI activation coordinates from tables in the corresponding article text. These term to activation mappings are used to construct the database. Automated meta-analysis is performed for a psychological term of interest  to construct a whole-brain association test map of the posterior probability of a term of interest occurring given activation at each voxel. This contrasts with forward inference maps such that are commonly obtained in task-based fMRI, or conventional meta-analyses, which often display the probability of brain activation given a task, or term. Therefore, association test maps may be conceptualized as meta-analytic maps identifying brain location activations, that are relatively more selective for the neurocognitive function of interest than forward inference maps. This procedure controls for the fact that many brain locations are implicated in multiple functions and are non-specifically activated in experiments. The process by which maps are generated by Neurosynth is wholly automated, and multiple validation techniques were applied by the original authors to compare results with manual techniques  in the brain where individual effect maps were spatially coincident with activations in association test maps for each neurocognitive function. This code is available in GitHub at ninadelacy/effect-mapping. Every combination of significant F > M and M > F effects and association test maps was computed, to determine voxels that were present in both maps for each combination. To create aggregated maps of effects in each subject across each neurocognitive function, overlapping voxels from F > M and M > F were collected, and redundancies eliminated to determine only unique voxels. We calculated the relative numbers of brain locations implicated in each neurocognitive functional map for F > M and M > F by summing the unique voxels for each neurocognitive functional map and dividing into the relative proportions for each subject group.We determined the spatial location of significant sex-related effects in each of the 8-, 24-, and 51-network models .Our analyses revealed that significant  sex-related differences were widely present in intrinsic networks in each of the three models , 4, rangOf note, occasional differences were observed among networks with similar neurocognitive functions. For example, among the speech and language group, we identified only F > M effects in an IN associated with language comprehension in the 24-network model, but both M > F and F > M effects in a similar IN in the 51-network model. In the motion-matched sample these asymmetries were typically reduced given the increased number of sex-related effects present.Analyzing 3 model orders permitted the examination of sex-related effects in brain networks in increasing detail. For example, while one sensorimotor network was present in the 8-network model, 9 sensorimotor networks were present in the 51-network model, associated with differing functional emphases. A consistent finding was that both M > F and F > M effects were present in all default mode networks, regardless of model order , 4. The The size of sex-related effects varied across individual INs, as did the spatial area of each network that displayed dimorphism. As may be appreciated in We also observed that M > F effects consistently occupied a larger average proportion of network territory than F > M. This phenomenon was replicated across all models in both the original and motion-matched samples –5. The dWe computed the proportion of total locations with dimorphic effects accounted for by each network . CompariWe computed the spatial overlap between unique locations with significant dimorphic effects  and association test maps for individual neurocognitive functions that have been most frequently associated with sex-related differences in task performance and in a set of cognitive control functions. In both sets of functions, the sex-related neurocognitive footprint was consistently and substantially larger in males than females, across all tasks and model orders . In the These findings were replicated in the motion-matched sample, where the number of unique dimorphic locations overlapping neurocognitive association test maps was increased. Typically, ∼200 more overlapping dimorphic locations overlapped neurocognitive maps in the 8- and 24-network models, and ∼400 more in the 51-network model . Of note•Sexual dimorphism was present in the majority of intrinsic functional networks in a large group of neurotypical young adults, regardless of model order.•The proportion of intrinsic networks with significant sex-related differences in activity ranged from 60 to 80% of all networks tested over 3 models of whole brain function.•Significant sex-related effects were present in all sub-networks of the default mode network group in all 3 models. This contrasted with other network groupings  where sex-related effects were less pervasive•On average, the effect size of significant sex-related differences in intrinsic network maps was larger in females, but occupied a greater spatial proportion of the network in males. The size of sex-related effects and proportion of network affected did not appear to be related.•Control networks (default mode and task-positive intrinsic networks) and the cerebellar networks contained the largest numbers of individual brain locations (voxels) with significant sex-related differences in activity•While our major results were replicated in the motion-matched sample, we observed a greater number of individual brain locations with significant sex-related differences in the smaller motion-matched sample of 535 subjects vs. the sample of 670 subjects matched for age and sex, but not head motion.a posteriori assignment of nodes to network identifications in youth (average age 15 years), dimorphism was also detected by another group in a majority of intrinsic networks  by networks . We provper se), and in the default mode networks. In the latter, we detected dimorphism across every sub-network, even as the overall default mode system split into increasingly smaller networks in the 51-network model, with some indication that posterior components of the system had larger effect sizes. Control networks – especially the DMN – dominated the spatial extent of sex-related effects, consistently occupying the lion’s share (>50%) of dimorphic locations in all models. The cerebellum was also highlighted. Our results suggest that the function of control networks and the cerebellum, especially the default mode system, may be most strongly influenced by sex in comparison to other network types. Our work therefore disagrees with smaller studies that failed to detect sex-related differences in default mode and control network function  and female  performance and also in a set of cognitive control functions where there has been much less consistent evidence of sex-related differences in performance. While our analytic strategy does not impute causation, this finding does suggest that cognitive task performance is not simply associated with the spatial extent of dimorphism in intrinsic networks, since the larger footprint of M > F effects was present in cognitive functions where both superior and inferior performance has been observed in males and in a range of cognitive control functions where sex-related performance differences are less robustly found. Rather, it may link to prior work suggesting males and females recruit different brain regions to accomplish similar tasks, which would also be consistent with our finding of a mosaic of M > F and F > M effects within the same networks. For example, An important result of our study, which provides the first whole-brain surveys of dimorphism with simultaneous multilevel modeling of brain functional networks, is that sex becomes a more influential variable as increasingly detailed functional parcellations are formulated. Previous work suggests that higher model orders constructed using ICA produce increasingly refined networks and it is also reasonable to suppose that as model order increases, larger networks split into subnetworks with more closely delineated functional associations. For example, work by p = 9.365 × 10-18). In both cases higher levels of head motion obtained in males, particularly younger adult subjects. It is also notable that historically, higher levels of head motion have been detected in subjects with conditions such as autism and ADHD, which are more common in males, as well as in younger subjects more generally. Indeed, while efforts are increasingly devoted to identifying sources of disparity in head motion in brain imaging research, and eliminating and controlling for the effects of motion, the relationship between sex and head motion has not been extensively explored. While further studies will inform the generalizability of our findings, the current study suggests the relationship between sex and motion should be carefully accounted for in functional brain imaging studies.While in general our effects were replicated in a motion-matched sample, motion may still play an important role and should always be carefully considered in fMRI studies. We found a significant difference in head motion as measured using DVARS between males and females after matching for age and sex in a sample of 670 neurotypical young adults. In the foundational dataset used for the present study, where imaging is available from a larger sample of 1570 young adults (not matched for age or sex), this was also the case (Limitations exist in the present study. Firstly, IQ scores derived from the Shipley-Hartford Age-Corrected T-scores were provided in the original dataset and used in the current analysis, that are estimates of IQ rather than the result of full IQ testing, and as may be seen in Using a multilevel modeling strategy to survey sex-related differences in intrinsic functional networks in increasing model orders of 8-, 24-, and 51-network models derived from whole-brain imaging, we identified a mosaic of sex-related effects in the majority of networks, affecting in total a small proportion of ∼0.5–2% of all brain locations. Dimorphism proved most prominent in the control networks and cerebellum, being particularly pervasive in the DMN system, with a much larger neurocognitive footprint in males. We conclude that modeling sex as a biological variable and as a covariate in analyses of human brain function using rsfMRI is required, and that high-order models which include a greater number of networks and/or more detailed functional parcellations are likely to be even more sensitive to sex-related differences. Our results extend prior investigations using task fMRI to provide additional support derived from intrinsic brain function measured in the resting state to suggest that males and females may recruit different spatial locations and proportions of brain networks to perform similar neurocognitive tasks. We highlight that our results pertain to brain function in a single, albeit comparatively large, cross-sectional sample of young adults. No conclusions can be drawn as to the causes or dynamic evolution of observed sex-related network differences in this group. The development of neural functional connectivity is a dynamic process with considerable reorganization and resculpting observed during maturation through young adulthood . Many biThe present study uses secondary data and was deemed not human subjects research by the University of Washington Institutional Review Board.NdL designed the study, performed the analysis, and wrote the manuscript. VC helped design the study and contributed to writing the manuscript. JK and EM contributed to study design and writing the 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.


Objective: This study aimed to determine the extent to which robotic arm rehabilitation for chronic stroke may promote recovery of speech and language function in individuals with aphasia.Methods: We prospectively enrolled 17 individuals from a hemiparesis rehabilitation study pairing intensive robot assisted therapy with sham or active tDCS and evaluated their speech (N = 17) and language (N = 9) performance before and after a 12-week (36 session) treatment regimen. Performance changes were evaluated with paired t-tests comparing pre- and post-test measures. There was no speech therapy included in the treatment protocol.Results: Overall, the individuals significantly improved on measures of motor speech production from pre-test to post-test. Of the subset who performed language testing (N = 9), overall aphasia severity on a standardized aphasia battery improved from pre-test baseline to post-test. Active tDCS was not associated with greater gains than sham tDCS.Conclusions: This work indicates the importance of considering approaches to stroke rehabilitation across different domains of impairment, and warrants additional exploration of the possibility that robotic arm motor treatment may enhance rehabilitation for speech and language outcomes. Further investigation into the role of tDCS in the relationship of limb and speech/language rehabilitation is required, as active tDCS did not increase improvements over sham tDCS. Following stroke, participation in professional, social, and family environments is often limited by acquired deficits affecting motor control, sensation, cognition, and communication. Treatment in each domain can promote some limited recovery, but residual disability remains a problem. Thus, there is a need for efficient interventions to expedite and enhance recovery. We explored the possibility that treatment in one domain—motor function—may benefit performance in another, untreated domain—communication—in individuals with acquired aphasia and/or apraxia of speech. Synergistic effects of treatment across domains could provide a transformational approach to stroke rehabilitation.The possibility that robotic motor treatment preceded by tDCS may benefit speech-language recovery was raised by Hesse et al. . They reN = 11) because the normative samples for the measures given are based on native English speakers. Two English speakers were unable to complete the full battery at post-test, leaving complete language data sets for 9 people for the Western Aphasia Battery-R and 10 people for the category naming task. Speech motor control data for 17 participants is reported here. All participants completed the entire 36 session hemiparesis protocol.A subset of seventeen participants  from a large multi-site RCT were enrolled in the speech and language study reported here (dates: 4/2013-9/2014). All treatment and assessments were conducted at Burke Medical Research Institute and Feinstein Institute for Medical Research with participants consented under IRB approval of both institutions. Eligibility criteria for the larger RCT were: a single, left-hemisphere ischemic lesion; cognitive function sufficient to understand the instructions and task in the study; and a Motor Power score ranging from 1 to 4/5, indicating that participants were neither hemiplegic nor had fully recovered motor function in the muscles of the shoulder, elbow, and/or wrist. Participants in the speech and language subset also presented with chronic aphasia and/or apraxia of speech subsequent to the same lesion. Speech-language testing occurred prior to the beginning of motor therapy (pre-test baseline) and again after the conclusion of the therapy sessions (post-test). All 17 speech-language participants were administered a speech motor control task . Language testing was only conducted on individuals whose primary language was English (Diadochokinetic rate (DDK) assesses the ability to produce sequential and alternating syllables . We used the test version from the Apraxia Battery for Adults 2nd version (ABA-2) . This frequently used verbal fluency measure requires lexical access and word retrieval . UnimpaiThe Western Aphasia Battery-Revised (WAB-R)  is a com2) surrounded by saline-soaked sponges. The center of the anode was placed 5 cm lateral to the vertex, and the cathode was placed over the right supraorbital region. For participants in the sham tDCS condition, current was ramped up to 2 mA over 30 s and then ramped down to 0 ms (All participants were enrolled in the double-blind repetitive right upper-extremity motor therapy study that included 36 sessions (3x/week) of robotic arm motor therapy (~1 h each) preceded by 20 min of tDCS. In this study, a 2 mA current was delivered by a battery-driven current generator using surface rubber-carbon electrodes . Participants were assessed during the week prior to the first treatment session (pre-test baseline), and within 5 days of the last treatment session (post-test). The same SLP administered baseline and post-test evaluations for each participant. Participants did not receive speech-language therapy as part of the treatment.t-test to evaluate overall change from baseline to post-test; and an independent samples t-test using the difference scores for each participant to evaluate tDCS (active~sham) group differences. Despite the small sample size, we use t-tests to allow for confidence intervals to get a sense of the effect size, and we also report the outcomes of non-parametric Wilcoxon tests throughout the results.To evaluate changes, we performed two analyses for each speech-language measure: a within-subjects paired t-test: p < 0.001; Non parametric Wilcoxon: p < 0.001]. As in the full cohort from the robotic arm study, improvement did not differ between tDCS and sham arms in the full set of 17 participants  or in the subset with WAB data discussed below .Demographic and stroke information, as well as tests they completed and tDCS group assignment, are presented in Table t-test revealed significant improvement in DDK scores from baseline to post-test .A paired t-test revealed a significant increase in category naming scores from baseline to post-test overall .A paired t-test revealed significant improvement in the WAB-R AQ from baseline to post-test .A paired In our data, chronic stroke participants with speech and/or language impairment exhibited detectable improvement on speech-language measures following intensive robotic arm rehabilitation preceded by tDCS. Critically, this improvement was observed in the absence of speech-language therapy, suggesting the possibility of synergistic effects across these distinct domains of stroke recovery. It is worth noting that Meinzer et al.  reportedt-test (but not a non-parametric test). This result leaves open the possibility that active tDCS was actively detrimental to improvement on this task, although it is surprising that this finding would occur for only one measure. We also note that tDCS groups were not matched for speech-language ability as part of the RCT and they were not matched in the study. The small sample size here also precludes us from determining whether other demographic or neurological factors  with electroencephalography (EEG) and magnetic resonance imaging (MRI). The remaining 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.


Identification of participants at clinical high-risk (CHR) for the development of psychosis is an important objective of current preventive efforts in mental health research. However, the utility of using web-based screening approaches to detect CHR participants at the population level has not been investigated.n = 356 interviews were conducted (response rate: 19.9%) using the Comprehensive Assessment of At-Risk Mental State (CAARMS) and the Schizophrenia Proneness Interview, Adult Version (SPI-A). n = 101 CHR participants and n = 8 first-episode psychosis (FEP) were detected. ROC curve analysis revealed good to moderate sensitivity and specificity for predicting CHR status based on online results for both UHR and BS criteria . Selection of a subset of 10 items from both PQ-16 and PCA lead to an improved of specificity of 57% while only marginally affecting sensitivity (81%). CHR participants were characterized by similar levels of functioning and neurocognitive deficits as clinically identified CHR groups.We tested a web-based screening approach to identify CHR individuals. Potential participants were invited to a website via e-mail invitations, flyers, and invitation letters involving both the general population and mental health services. Two thousand two hundred seventy-nine participants completed the 16-item version of the prodromal questionnaire (PQ-16) and a 9-item questionnaire of perceptual and cognitive aberrations (PCA) for the assessment of basic symptoms (BS) online. 52.3% of participants met a priori cut-off criteria for the PQ and 73.6% for PCA items online. One thousand seven hundred eighty-seven participants were invited for a clinical interview and These data provide evidence for the possibility to identify CHR participants through population-based web screening. This could be an important strategy for early intervention and diagnosis of psychotic disorders. In parallel, clinical high-risk (CHR) criteria also have been based on the basic symptom (BS) concept8 that are based on the presence of self-experienced perceptual and cognitive anomalies that are thought to represent the earliest manifestation of psychosis risk.8The identification of young people at high-risk for the development of severe mental health disorders is an important objective in current research and clinical practice.9 with transition rates ranging between 10% and 50% over a 2- to 5-year period.10 Interestingly, recent studies have shown that the combined presence of both BS and UHR criteria increases the predictive power significantly.11 The importance of detecting CHR individuals is also underlined by the fact that at-risk populations show high correlations with nonpsychotic symptoms12 and mental health outcomes in those cases that do not develop psychotic disorders meet criteria for a range of other diagnoses, including affective disorders, personality disorders, and substance abuse.14There is now consistent evidence that these instruments allow a reliable identification of CHR participantsA major limitation for current approaches to detect CHR participants is the fact that CHR criteria need to be established through semi-structured interviews administered by trained personnel in help-seeking populations who are already within the health care system. Accordingly, it would be important to develop novel ways of identifying CHR participants that could potentially circumvent clinical entry points and allow population-wide screening of potential signs of impending psychosis and related mental disorders.http://www.your-study.org.uk which includes an initial screening through the 16-item version of the prodromal questionnaire (PQ-16)15 and a 9-item scale of perceptual and cognitive aberrations (PCA). Previous studies using questionnaire-based screenings in mental health settings and the general population highlighted the potential utility of using screening tools to identify CHR individuals.16–18 For example, the PQ-16 produced correct classification of UHR individuals in a secondary mental health setting with high sensitivity and specificity.15E-mental health applications, such as online screening for emerging mental health conditions, could provide an important approach to target young people at-risk for psychosis as they could facilitate identifying potential participants who would benefit from more detailed psychiatric assessments. To investigate this possibility, the Youth Mental Health Risk and Resilience Study (YouR Study) implemented a website Accordingly, we tried to determine whether questionnaire-based assessments of both attenuated psychotic experiences and BS through an online platform are characterized by sufficient sensitivity and specificity to predict CHR status as determined by clinical interviews. A secondary objective was to characterize the neurocognitive and psychopathological characteristics of CHR participants recruited through an online approach and compare results with at-risk samples in previous studies that were identified through clinical pathways.Specifically, we predicted that self-reported levels of attenuated psychotic experiences and BS obtained online would predict CHR criteria as assessed through the CAARMS and SPI-A interviews. In addition, CHR participants identified through online screening were expected to be characterized by similar levels of functioning and neurocognitive deficits as clinically identified CHR individuals, suggesting that E-mental health applications may be effective in the detection of early signs of psychosis in the general population and in help-seeking participants.19 The YouR study is funded by the Medical Research Council (MRC).The YouR Study is a cross-sectional study to identify neurobiological mechanisms and predictors of psychosis-risk with a state-of-the-art neuroimaging approach  in combination with core psychological processes, such as affect regulation, adverse experiences, and attachment.http://www.your-study.org.uk as part of the study protocol and potential participants were invited through e-mail invitations, flyers, advertisements, and general practioners (GP) letters. Specifically, email invitations were sent out to colleges and Universities in Glasgow and Edinburgh. In addition, posters and flyers were advertised in NHS clinics and public transportation. Finally, databases of GP practices were searched for potential participants who then received an invitation letter that directed them to the website.A website was implemented The study was approved by the ethical committees of the NHS Research Ethical Committee Glasgow & Greater Clyde.a) the PQ-1615 and (b) a 9-item PCA scale that was developed to assess BS 20 in a help-seeking population attending secondary mental health services. A cut-off score of 6 or more items was associated with UHR criteria as assessed by the CAARMS with high sensitivity (87%) and specificity (87%). Items for the PCA were generated from existing patient descriptions of cognitive and perceptual experiences21 and from the SPI-A.22 Participants were asked to provide ratings based on experience in the last 12 months.Informed consent for the web screening was provided online, followed by 2 questionnaires: 22 to determine at-risk criteria as defined by cognitive-perceptive basic symptoms (COPER) and cognitive disturbances (COGDIS) were administered through trained research assistants and MSc/PhD level researchers. CHR participants were excluded for current or past diagnosis with Axis I psychotic disorders. Other co-morbid Axis I diagnoses, such as mood or anxiety disorders, were not exclusionary and all participants were between 16 and 35 years of age .Participants who met online inclusion criteria  were invited through an email to participate in the second part of the study that involved clinical assessments to determine CHR status and neuropsychology assessments . At the 19 Moreover, the scales for premorbid adjustment,23 social and functional role scales24 and the Brief Assessment of Cognition in Schizophrenia Battery (BACS) were administered.25 Participants were recruited into the CHR group if they meet (a) SPI-A COGDIS/COPER criteria, (b) ARMS attenuated psychosis group (subthreshold psychotic syndrome present in the last year without a decline in functioning), (c) ARMS vulnerability group , and (d) ARMS BLIPs group (brief limited intermittent psychotic symptoms).A second appointment served to verify CAARMS and SPI-A items and the M.I.N.I. International Neuropsychiatric Interview (M.I.N.I. 6.0).Data were analyzed using SPSS version 18.0.To investigate the sensitivity and specificity of online screening scores toward predicting CHR status, we conducted ROC analyses. Pairwise comparison of ROC curves was performed to test the statistical significance of the difference between the areas under the ROC curves of the PQ-16 and the PCA. The accuracy of the test depends on how well the test separates the group being tested into those with and without the disorder in question and is measured by the area under the curve (AUC). An AUC test value equal to 0.5 means that there is no difference and when the value equals 1 there is perfect separation of the values of the 2 groups.n = 2296 participants completed the online screening from the commencement of the study in September 2014 until July 2017. 52.3% participants (n = 1202) fulfilled the PQ-16 cut-off criteria (≥6 or more items) while 73.6% (n = 1691) met criteria for the PCA (≥3 or more items). Participants were invited for interviews until July 2017 and 356 interviews were conducted (a response rate of 19.9%). There were no significant differences between participants who accepted invitations for interviews vs those who did not on the PQ-16 . A statistical trend emerged for higher scores on the PCA scale for participants who attended clinical assessments .A total of n = 101 participants met CHR criteria and n = 8 first-episode (FEP) criteria : 1, bipolar disorder with psychotic features: 1). FEP was defined as a person who fulfilled DSM-V for psychotic disorders that had previously not been in contact with mental health services. N = 215 were below CHR criteria (CHR negative group). For n = 32 participants, there was insufficient data to determine CHR status due to withdrawal from the interview and/or the tattoos/piercings that were exclusion criteria of the neuroimaging protocol. These participants were excluded from subsequent analysis. For 14 CHR participants, neuropsychological data were not available. Of the 101 CHR participants, n = 87 were recruited from the general population  while 14 were recruited through the National Health Service (NHS) .CAARMS/SPI-A interviews were conducted and n = 27 participants who scored below online cut-offs for PQ and PC-Q items . Two out of 27 participants met CHR criteria. However, there was a significant delay between the initial online score and clinical assessments, suggesting that CHR symptoms emerged after the entry of questionnaire data.In addition, we carried out SPI-A and CAAMRs interviews in n = 8; other services : n = 7). Thirty-one CHR participants disclosed no previous or current contact with mental health services while 55 had previous contact . For 10 CHR participants, no information was available.Demographic and clinical characteristics of CHR participants, CHR negative, OVS, and control groups as well as a sample of matched control participants are summarized in CHR participants were characterized by a significant lower GAF score compared with CHR negative participants. The OVS group was significantly younger than CHR negative participants and had fewer male participants and had less years of education.Compared with controls, the CHR group was characterized by reduced role and social functioning as well as impaired premorbid adjustment. The large majority of CHR participants met also criteria for Axis-1 affective and anxiety disorders . CompariCronbach’s alpha was measured to assess the internal consistency of the PCA questionnaire. The total score on PCA was associated with a value of 0.786, suggesting that the items have high internal consistency.n = 27 participants that did not meet online cut-off criteria   .The AUC for the PQ-16 for predicting CAAMRS status was significant indicating that total symptom endorsement score was predictive of CAARMS status above chance level. A cut-off score of 6 or more symptom items on the PQ-16 was found to identify at-risk status with a sensitivity of 81% and specificity of 44%. Increasing the cut-off score to 7 endorsed items decreased sensitivity to 73% but increased specificity 55%.The AUC for the PCA was also significant . A cut-oTo assess the utility of combining the subscales of the online questionnaire to predict CHR status further ROC analysis was conducted where the PQ-16 and PCA subscales were combined . The AUC26 which estimates the utility of features to a classification problem in the situation where features may have conditional dependencies. For this analysis, we selected all 25 items from both the PQ-16 and the PCA in addition to 3 demographical variables: (a) age, (b) sex, and (c) familial risk (first degree relative with a psychotic disorder) that were obtained during the initial clinical assessment. We took the average predictive weights from 10 samples of 234 individuals’ data set and ranked the features  that optimized the likelihood ratio (LR+) that included familial risk, improved specificity (57%), while only marginally affecting sensitivity (81%).The current study examined the possibility of detecting CHR participants through a web-based screening approach that utilized the PQ-16 as well as a 9-item PCA scale in the general population and through clinical services. Our data suggest that both screening instruments are characterized by good to moderate sensitivity and specificity to predict CHR status as defined by CAARMS and SPI-A interviews.27 The online implementation of the PQ-16 in combination with a previously established cut-off 6 or more items yielded similar sensitivity and specificity values to predict UHR status established through a semi-structured interview as in the study by Ising et al.15 Specifically, we observed that a score of 6 or more positively answered items was associated with UHR status as assessed by the CAARMS with good sensitivity (81%) and specificity (44%).The PQ and abbreviated versions have been used in previous studies to detect CHR participants in secondary mental health settings.11 Currently, it is unclear whether BS can be reliably predicted from self-report measures. The 9-item PCA was characterized by excellent sensitivity for predicting COPER and COGDIS status assessed through the SPI-A interview (95%) but low specificity (13%). Our data show that changing the cut-off to 4 or more items was associated with improved specificity.A novel approach of the current study was the implementation of a screening instrument to predict self-experienced PCAs that are highly predictive for later transitioning to psychosis.The combination of a subset of features from the PQ-16 and PCA that included familial risk furthermore improved specificity (57%), highlighting the potential for improved online screening using a novel combination of features. However, we wish to note that this screener would require further validation in an independent sample.28An important issue concerns the clinical and demographic characteristics of CHR participants in the current sample who are self-referred and identified through an online screening from the general population and clinical services. Overall, the majority of CHR participants were recruited from the general population and only a minority were in current or past treatment with NHS services. Our data indicate that the current sample shares similarities with existing CHR populations identified through clinical referral pathways. Specifically, the CHR participants identified through our web interface are characterized by reduced GAF scores and impaired role and social functioning. However, especially role and social functioning was somewhat higher than in previous CHR samples.29 we observed mild impairments in neurocognition in CHR participants compared with healthy controls. Specifically, reductions on the digit symbol and motor token task were observed that replicate findings highlighting the contribution of reductions in processing speed toward psychosis risk.31 Interestingly, both verbal memory and executive functions were found largely to be intact in the current CHR sample that is in contrast to deficits reported in previous studies.33Consistent with previous data,34 Together with the presence of PTSD and eating disorders in the current sample, these findings support the view that CHR participants express a high degree of comorbidity with other psychiatric syndromes12–14 which are likely to impact on the trajectory and functional outcome.35Baseline assessment of current psychopathology furthermore revealed extensive psychiatric co-morbidity of CHR participants. In particular, both anxiety and mood disorders were present in the large majority of CHR participants consistent with previous formulations that that changes in affect are frequent early signs before the onset of psychosis.n = 27 participants, the relationship between scores below the online cut-off for CHR status and SPI-A as well as CAARMS criteria. Finally, since both PQ-16 and PCA measures were completed online, it is possible there was an overendorsement of PLE and BS . Moreover, it was not possible to determine how many of the target population visited our website. However, we would like to note that the frequency of both PLEs as well as CHR criteria were significantly less prevalent in our online sample compared to estimates obtained from the general population,38 suggesting that the help-seeking participants were self-selecting with significantly higher levels of PLE as well as affective disorders.The current findings have a number of limitations. Firstly, we could only verify at-risk criteria established through online screening for UHR and BS symptoms in a subgroup of individuals as only 19.9% of those participants who met online screening cut-offs were evaluated through CAARMS and SPI-A interviews. However, the current data suggest that there were no significant differences in scores on the PQ-16 and only a trend for difference PCA scores. Secondly, we could only establish in a small sample of In the current sample, ~1 out of 3 participants who met online cut-off criteria was also found to either meet UHR or SPI-A status, suggesting that population wide-screening may be useful in the detection of emerging signs of psychosis and related mental health outcomes. In addition, we also would like to note that the current recruitment approach identified a significant number of participants meeting criteria for FEP, further highlighting the potential of an online screening for identifying young people that are at-risk for serious mental health problems.39 Together with the modest specificity of our online screening, this raises the issue of a substantial number of false positive and the risk of conferring stigma in detecting CHR participants.40 We would like to note, however, that in the course of interviewing a large number of CHR participants recruited through our online pathway, we rarely encountered concerns about being labeled. This anecdotal observation is consistent with data suggesting that young people who are deemed to be at potential risk for psychosis value the opportunity of talking about their experiences rather than express explicit concerns about stigma.41However, several issues need to be addressed in future studies to improve the clinical utility of an E-mental health approach to detect emerging psychosis. Firstly, follow-up data on the current sample will provide further information on transition rates of CHR participants identified through online screening. There is emerging evidence that at-risk samples recruited primarily through outreach activities may be characterized by lower transition rates than in samples referred through clinical pathways.43 This is supported by emerging evidence suggests that additional clinical and demographic variables increase accuracy of predicting conversion to psychosis in clinically ascertained CHR samples.43 Such refined online screening could also potentially allow the detection of FEP cases as demonstrated by findings from the PQ-16 in our sample that showed that the FEP group had significantly elevated scores compared to CHR participants. This issue is potentially important as current evidence suggests that specialized high-risk clinics only identify around 5% of FEP cases.42 Accordingly, development of novel tools that allow the identification of both FEP cases as well as participants meeting CHR criteria would be important for early intervention.Nonetheless, it is important that future studies investigate the possibility of improving sensitivity and specificity of online detection of CHR participants through additional variables, such as premorbid adjustment, psychosocial functioning and neurocognition, that are associated with the CHR status and psychosis risk.In summary, our data provide evidence that it is possible to identify CHR participants through online screening at the population level. This approach could have implications for efforts to identify young people at-risk of mental health problems as well as for the identification of emerging psychosis. Future studies are required to improve sensitivity and specificity through the addition of clinical, demographic, and neurocognitive variables. Moreover, it is conceivable that the current framework could be extended to secondary mental health settings as well as for related disorders that typically emerge during the transition from adolescence to adulthood, such as affective disorders and borderline personality disorder.This study was supported by the project MR/L011689/1 from the Medical Research Council (MRC).Supplementary Figure 1Click here for additional data file.


DNAzymes, derived by in vitro selection processes are also able to bind and cleave RNA targets and therefore down-regulate gene expression. The purpose of this review is to present and discuss several applications of ribozymes and DNAzymes in muscle and brain. There are several diseases which affect muscle and brain and catalytic nucleic acids have been used as tools to target specific cellular transcripts involved in these groups of diseases.The discovery of catalytic nucleic acids (CNAs) has provided scientists with valuable tools for the identification of new therapies for several untreated diseases through down regulation or modulation of endogenous gene expression involved in these ailments. These CNAs aim either towards the elimination or repair of pathological gene expression. Ribozymes, a class of CNAs, can be mostly used to down-regulate (by RNA cleavage) or repair (by RNA  One of the most characterized DNAzyme is the 10-23 subtype consisting of a cation-dependent catalytic core of 15 deoxyribonucleotides that binds to and cleaves its target RNA between an unpaired purine and paired pyrimidine through a de-esterification reaction [in vitro and in cell culture systems. A wide range of chemical modifications has allowed the synthesis of oligonucleotides with in vivo stability approaching that of most conventional drugs [Selective gene silencing by catalytic nucleic acids is a field that has been used with great success for studying natural processes in muscle and brain. Furthermore, these catalytic nucleic acids have been widely used for establishing novel gene therapy approaches for many muscle and brain diseases. In most cases, catalytic nucleic acids aim towards either the elimination or repair of pathological transcripts. Catalytic nucleic acids can be separated into two main categories: ribozymes and DNAzymes. Ribozymes, the most extensively studied of the catalytic nucleic acids, exist in a range of distinct categories of naturally occurring catalytic RNA. These include a series of small ribozymes important for the rolling circle replication of viroid genomes, such as hammerhead and hairpin ribozymes ,2,3, groolecules . DNAzymereaction . The catal drugs .in vivo remains a critical challenge for successful transition from the laboratory to the clinic. Beside ribozymes and DNAzymes, advances in molecular genetics have revealed new tools for selective gene silencing. Small interfering RNAs (siRNAs) are single strands of RNA, approximately 20 nucleotides long, which are able to target, cleave and inactivate specific mRNAs . This poin vitro and in vivo [Beside siRNAs, antisense oligonucleotides have been used to inhibit gene expression levels both  in vivo . Antisen in vivo . During Ribozymes and DNAzymes have been widely used towards the better understanding and therapy of various skeletal and smooth muscle disorders. Myotonic dystrophy type I, myotonia congenita and neuromuscular junction abnormalities, restenosis of coronary arteries and hypertension are some of the disorders that scientists introduced the use of ribozymes and DNAzymes . In this review, we will focus on different strategies implicating the function of ribozymes and DNAzymes in the pathomechanisms and therapy of these disorders . The vascular smooth muscle cell in mature animals is a highly specialized cell whose principal function is contraction and regulation of blood vessel tone-diameter, blood pressure, and blood flow distribution. However, abnormal environmental signals can lead to adverse phenotypic switching and acquisition of characteristics of smooth muscle cell that can contribute to the development and/or progression of vascular disease ,20. SeveAngiotensin II (Ang II) plays an important role in the development of hypertension and atherosclerosis by inducing vascular smooth muscle cell growth and synthesis of aldosterone ,22. Actiet al. described the activity of several ribozymes targeting c-myb mRNA. Hammerhead ribozymes were capable of cleaving c-myb RNA and as a result inhibit smooth muscle cell proliferation [et al. explored the use of hammerhead ribozymes as tools to knock down specific activators of cell proliferation. They designed two specific hammerhead ribozymes to inhibit the mRNA levels of cyclin E and E2F1, two potent activators of cell proliferation which cooperate to promote the G1 to S phase transition [Proliferation of injured smooth muscle cells contributes to the reocclusion or restenosis of coronary arteries that often occurs following angioplasty procedures. Coronary angioplasty is an effective procedure used in order to open occluded vessels. However, in spite of a number of technical improvements in the procedure, post-operative occlusion of arteries, or restenosis, still occurs. It is widely believed that by preventing the injury-induced activation and proliferation of medial smooth muscle cells after angioplasty, intiminal thickening and restenosis could be prevented . In an aferation . This fiansition ,28. Afteansition . et al. identified most of the accessible ribozyme target sites in the 3’ UTR of the DMPK mRNA and designed a hammerhead ribozyme to cut the most accessible site. The use of these hammerhead ribozyme significantly reduced the number of mutant DMPK mRNA-containing nuclear foci in human DM1 myoblasts. The reduction of mutant DMPK mRNA and nuclear foci had as an effect the partial restoration of insulin receptor isoform B expression in DM1 myoblasts [Ribozymes and DNAzymes have been also used as tools to target defects in neuromuscular diseases. Myotonic Dystrophy type 1 (DM1) is a degenerative neuromuscular disease characterised by a large CTG repeat expansion situated in the 3’ UTR of the DMPK gene. The mutant DMPK 3’ UTR RNA containing the CUG expansion accumulate to form RNA foci in the nucleus of DM1 cells ,31. Thesyoblasts . et al. designed a group I intron ribozyme in order to modify the CUG repeat expansion at the 3’ UTR of the human DMPK transcripts. This group I intron ribozyme showed to be capable of ligating a small mRNA fragment, contained within the ribozyme, to a simple DMPK-target RNA in vitro. It also modified a larger target transcript, leading to replacement of twelve repeats with five repeats, both in vitro and in mammalian cells [Another approach was the use of specific ribozymes for the repair of the expanded repeat. Phylactou an cells . et al. designed a modified Tetrahymena ribozyme to mediate trans-splicing repair of the mutant canine skeletal muscle chloride channel (cClC-1) mRNA transcripts. The ribozyme was able to target the mutant mRNA and replace the mutant containing 3’ portion by trans-splicing the corresponding wild type sequence. Furthermore, when the chloride channel function was examined in single cells, a wide range of electrophysiological activity was observed, with 18% of cells exhibiting significant functional restoration and some cells exhibiting complete rescue of the biophysical phenotype [The mutant canine skeletal muscle chloride channel (cClC-1) mRNA transcript that causes the inherited disorder myotonia congenita, prompted scientists to further investigate the feasibility of RNA repair using specific ribozymes. Rogers henotype .et al. designed hammerhead ribozymes in order to target RNA transcripts from four different slow channel congenital myasthenic syndrome mutations. These hammerhead ribozymes were able to efficiently discriminate between mutant and wild type RNA transcripts that differ only by a single nucleotide substitution [The muscle acetylcholine receptor (AChR) is expressed at the neuromuscular junction, and plays the principal role in nerve to muscle signal transmission. A number of mutations have been characterised in the AChR -subunit gene which affect receptor function and give rise to slow channel congenital myasthenic syndrome ,38. Abdetitution . Furthertitution .et al. demonstrated the first effective destruction of the mutant huntingtin mRNA using a specific DNAzyme that was able to cleave the mutant huntingtin mRNA in a sequence-specific manner, that lead to significant reduction of mutant huntingtin protein expression in mammalian cells [The use of ribozyme and DNAzyme strategies were also introduced in various brain disorders . Huntingan cells . Therefoan cells ,44. et al. designed RNA-cleaving ribozymes in order to control the expression of β-secretase. The scientific findings demonstrated that these ribozymes were able to significantly inhibit β-secretase gene expression at both the mRNA and protein levels [Catalytic nucleic acids have been also used in Alzheimer’s disease (AD). AD is a neurodegenerative disorder involving the deposition of senile plaques in the brain. The plaques consist of aggregates of a 4 kDa Aβ-amyloid peptide (Aβ). The peptide is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases ,46. The et al. showed that an mRNA transcribed in dihydrolipoamide succinyltransferase (DLST) gene was significantly lower in the brain of AD patients compared to the controls [In another study, Kanamori controls . The trucontrols ,48.Data from another AD study implicated the double-stranded RNA dependent protein kinase (PKR) in disease progression . A libraet al. used adeno-associated virus (AAV) vector delivery of a-synuclein ribozyme in order to test its silencing effect on degenerating nigrostriatal neurons in a PD animal model, expressing the 1-methyl-4-phenylpyridinium (MPP+) [et al. designed a ribozyme against human a-synuclein gene expression and constructed a ribozyme-expressing rAAV vector (designated rAAV-SynRz). Co-transfection of rAAV-SynRz and rAAV-a-synuclein into HEK293 cells resulted in down-regulation of a-synuclein protein expression in vitro. rAAV-SynRz was then injected into the substantia nigra of MPP+-treated rats. Cell counts of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra revealed that rAAV-SynRz significantly protected TH-positive cells against apoptotic death, compared with untransfected mice or those which were injected with a control reporter vector [Parkinson’s disease (PD) is another neurodegenerative disorder which is characterized by selective degeneration of substantia nigra dopamigenic neurons and the presence of abnormal cytoplasmic aggregates of a-synuclein . Scientim (MPP+) . MPP+ ism (MPP+) . Hayashir vector . These rde novo. Glioblastomas are lethal and nearly all patients die within one year, despite treatment [Glioblastomas are the most frequent malignant brain tumors, which can progress from lower grade gliomas or arise reatment . Ribozymreatment ,56.et al. showed in two cell lines, U87 and T98G, that stable ribozyme-targeting leads to a robust reduction of PTN mRNA and protein levels. Further investigations showed that the reduction of PTN mRNA and protein levels results in decreased migration and colony formation in both glioblastoma cell lines [et al. used a subcutaneous tumor xenograft model in order to transfect specific ribozymes against both PTN and ALK, this had as a result the abolishment of tumor growth [The secreted growth factor pleiotrophin (PTN), also called heparin binding growth-associated molecule (HB-GAM), heparin affin regulatory peptide (HARP), heparin-binding growth factor 8, heparin-binding neurotrophic factor (HBNF), or osteoblast-specific protein-1 (OSF-1) is a 15.3-kDa developmentally regulated cytokine, which shows very limited expression in normal adult tissues, but is markedly upregulated in various primary human tumors and tumor cell lines ,57. Sciell lines . When thll lines . In addill lines . ALK wasll lines . Grzelinr growth . in vitro. It may well be able to serve as a tumor angiogenesis factor [et al. transfected human melanoma cells expressing high PTN levels, found to be able to metastasize from subcutaneous tumors to the lungs of experimental animals, with ribozymes in order to reduce PTN expression levels. The reduction of PTN mRNA levels showed not to affect the growth of the melanoma cells in vitro. In nude mice, however, tumor growth and angiogenesis were decreased. Moreover, apoptosis in the tumors was increased and the metastatic spread of tumors from the subcutaneous site to the lungs was prevented [Clinical and experimental evidence showed that the metastasis of malignant cells from a localized tumor is directly related to the number of microvessels in the primary tumor. Tumor angiogenesis is thought to be mediated by tumour-cell-derived growth factors. PTN can induce the release of active proteolytic enzymes from endothelial cells and induce tube formation of endothelial cells s factor . To testrevented . 2+-dependent enzymatic activity capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions [et al. showed that intra-cerebral administration of a poly-(G)(10)-tethered, phosphorothioated DNAzyme in JEV-infected mice led to more than 99.99% inhibition of virus replication in brain, resulting in a dose-dependent extended lifespan or complete recovery of the infected animals [Finally, DNAzymes were used against the Japanese encephalitis virus (JEV). JEV is an arthropod-borne flavivirus with a single-stranded RNA genome containing non-coding regions at its 5’ and 3’-ends . Non-codnditions . These Dnditions . Appaiah animals .To date, several different therapeutic attempts have been described as potential treatment approaches for many muscle and brain diseases. The use of catalytic nucleic acids and more specifically of ribozymes and DNAzymes against these diseases has shown some promise. Several approaches have already been described in the literature which reveal the potential of ribozymes and DNAzymes as tools for the study or therapy in muscle and brain diseases. The sequence-specific action which both of these categories of catalytic nucleic acids have against target RNA makes them flexible agents for the elimination or repair of pathological gene expression in muscle and brain cells. Particularly the trans-splicing ribozyme has the unique feature to repair mutant transcripts which are implicated in both groups of diseases. Naturally, more effort should be dedicated, particularly in the area of delivery of nucleic acids so that the effect of both ribozymes and DNAzymes is more efficient and specific.