Females with autism spectrum disorder have been reported to exhibit fewer and less severe restricted and repetitive behaviors and interests compared to males. This difference might indicate sex-specific alterations of brain networks involved in autism symptom domains, especially within cortico-striatal and sensory integration networks. This study used a well-controlled twin design to examine sex differences in brain anatomy in relation to repetitive behaviors.n = 150, 62 females, 88 males) enriched for autism spectrum disorder (n = 32), and other neurodevelopmental disorders (n = 32), we explored the association of restricted and repetitive behaviors and interests—operationalized by the Autism Diagnostic Interview-Revised (C domain) and the Social Responsiveness Scale-2 —with cortical volume, surface area and thickness of neocortical, sub-cortical, and cerebellar networks.In 75 twin pairs (Co-twin control analyses revealed within-pair associations between RRBI symptoms and increased thickness of the right intraparietal sulcus and reduced volume of the right orbital gyrus in females only, even though the mean number of RRBIs did not differ between the sexes. In a sub-sample of ASD-discordant pairs, increased thickness in association with RRBIs was found exclusively in females in the orbitofrontal regions, superior frontal gyrus, and intraparietal sulcus, while in males RRBIs tended to be associated with increased volume of the bilateral pallidum.However, due to a small sample size and the small difference in RRBI symptoms within pairs, the results of this exploratory study need to be interpreted with caution.Our findings suggest that structural alterations of fronto-parietal networks in association with RRBIs are found mostly in females, while striatal networks are more affected in males. These results endorse the importance of investigating sex differences in the neurobiology of autism symptoms, and indicate different etiological pathways underlying restricted and repetitive behaviors and interests in females and males. Autism spectrum disorder (ASD) is a neurodevelopmental condition of complex origin, defined by challenges in social communication and interaction alongside restricted and repetitive behaviors and interests (RRBIs) that cause significant impairment in daily life functioning . A markeSex differences in symptom presentation of ASD have predominantly been reported in the domain of RRBIs. Despite some inconsistencies, see, e.g., , most stPrevious studies have investigated sex differences in brain structure regardless of RRBI symptom severity, and found non-overlapping structural changes in males and females , alteredMore generally, ASD is associated with environmental, shared and non-shared, as well as genetic components which likely contribute to the heterogeneity in the etiology , 22. TheAs part of the Roots of Autism and ADHD Twin Study Sweden (RATSS) , the objn = 288 twins included in RATSS to date, N = 261 had completed MRI scanning, from which we only included same-sex pairs with high-quality image scans for both twins. These inclusion criteria resulted in a sample of 75 same-sex pairs , of which 44 were male pairs (mean age 15.9 years) and 31 female pairs (mean age 16.4 years), and 46 monozygotic and 29 dizygotic pairs. Zygosity was determined with DNA testing (using a 48 single-nucleotide polymorphisms panel . The diagnostic cut-off for presence of clinically relevant RRBIs indicating ASD on the total score ≥ 2 . T1-weighted acquisition was the first 7-min scan of a 50-min scanning protocol, preceded by a 5 to 7 min mock scan training for self-control of head movements. During the mock scan training participants were provided with feedback about excess head movement (1.5 mm in any direction), by automatically stopping the movie they were watching. Head movement reduced during the practice for most participants.T1-weighted images were acquired on a 3 Tesla MR750 GE scanner at the Karolinska Institutet MR center . The well-established standard pipeline was run on the original T1-weighted images [B = − 0.04, p < 0.001) and thus having less image quality . However, our estimate of interest, RRBI’s (ADI-R) did not predict data quality or movement scores either across  or within pairs . From the subjects that were excluded due to excess movement, the mean symptom level of RRBI’s from the ADI-R C domain was 0.94, i.e., they did not have more RRBI symptoms, and the mean age of this group was slightly younger, 15.28 years, compared to 16.11 in the included sample. Finally, to assess the impact of data quality on our outcome, we ran the main analyses, i.e., the association between RRBIs from the ADI- R C domain and brain structure within pairs, split by sex, also on a subsample consisting of those with QC1 (n = 70), which largely replicated our findings .Raw images were processed in Freesurfer 6  using FAST (FMRIB's Automated Segmentation Tool within FMRIB's Software Library) which also corrects for spatial intensity variation. Segmented images were warped to MNI space using non-linear registration FNIRT from FSL. GM and WM volumes for the somato-motor cerebellar region were extracted from the intersection between the somato-motor regions in Buckner’s 7-network functional atlas, which includes anatomical regions IV, V, VI, and VIIB of the cerebellum , and segRRBIs are presumed to rely on a wide network of regions involved in motor function and cognitive control of neocortical and subcortical areas, in particular cortico-striatal circuits . In the r-project.org/https://www.).All statistical analyses were performed in R  and Kruskal-Wallis tests for continuous variables . Some of the variables were not normally distributed; for consistency reasons, non-parametric tests were chosen for all tests. These tests yielded no between group significant differences  cross-validated the findings with the RRB subscale from SRS-2 and (2) tested the specificity of the findings to RRBIs by controlling for social cognition. Total brain volume was adjusted for when assessing cortical volume and surface area, but not thickness, and IQ was adjusted for in all models.For the main analyses, a twin/co-twin design was implemented to investigate the association between RRBI’s on a dimensional scale (predictor) and the anatomy (outcome) of the regions of interest while controlling for unmeasured confounding factors shared within twin pairs . MZ and DZ twins were collapsed in order to increase statistical power. Within-twin pair associations were estimated using a conditional linear regression model within the generalized estimating equations (GEE) framework, using the dergee package from R . Herein,Within-pair analyses were run in three sub-steps. First, the association between RRBIs and brain structure was assessed for males and females separately.2 tests for each ROI that was associated with RRBIs in either males or females. A significant difference on a Wald-test indicates that the estimate of the association was different for the sexes. By doing the interaction analysis in this way, we could allow for confounding covariates to differ between sexes.Next, to compare the association between symptoms and structure in males and females, we computed Wald χn = 6 pairs) or discordant (n = 20 pairs) for ASD diagnosis .Further, to test the robustness of the observed effects, models otherwise identical to the models of the main analyses were run with a different estimate of RRBIs, RRB subscale of the SRS-2, which addresses current as opposed to life-time symptoms. Finally, specificity of the results toward RRBIs were tested by adding different autism symptom domains as covariates in the model, including the social cognition subscale from SRS-2, and reciprocal social interaction domain from the ADI-R, to control for highly correlated symptoms that might have confounded the observed effects. Additional analyses were run to control for interaction effects between age and RRBIs on brain anatomy showing significant associations for the right postcentral gyrus, superior precentral sulcus, and superior parietal sulcus, i.e., areas which were not associated with RRBIs in our study  in males associated with RRBIs. A separate FDR-correction was performed on all p values for the same comparison but in females. It must be noted that the model included sex as a factor, that is, only one model was run including both sexes, even though a p value list of outcomes for each sex was generated, on which the FDR correction was performed. The total number of comparisons in the main analyses is 36 (18*2) cortical regions * 3 estimates  + 10 subcortical regions (volume) + 6 cerebellar regions  = 124 associations per sex. Post-hoc Wald-tests were performed in order to compare males to females only for those regions that were significantly associated with brain structure in either males or females. Therefore, no multiple comparison correction was conducted on these tests. Further post-hoc analyses that were preformed included 124 comparisons each per sex for associations between brain structural estimates with the RRB sub-scale of the SRS-2; the ADI-R sub-scores A  and C (RRBI); the SRS-2 subscales RRB and Social cognition, and finally the interaction between RRBI’s from ADIR and age. FDR correction was performed per estimate  per test. In addition, we calculated sex differences between demographic data, with a total number of six tests. Across and within subjects’ associations between the different variables also included six tests each. The behavior associations and sex difference calculations were descriptive in nature. Therefore, no multiple comparisons corrections were performed. Sample size of the present study was comparable to recently published twin studies using similar co-twin designs reporting medium to large effect size [All ect size , 48.. Atect size .Males and females did not differ on overall RRBI symptom severity, other autistic symptoms and traits and IQ. Further, no within pair differences between the sexes were observed for any of these variables Table .B = 0.037, q = 0.012) . Moreover, there were associations only at q < 0.1 in females between RRBI symptoms and increased thickness of the right orbital gyrus  and right inferior frontal orbital gyrus , and reduced surface area of the left superior frontal gyrus . Increased surface area of the right middle frontal gyrus in relation to more RRBIs was observed when using a threshold of q < 0.1 . In males, on the other hand, no within-pair associations between RRBIs and brain anatomy were observed at any threshold. We did not observe any significant associations between subcortical or cerebellar regions and RRBIs in either sex.Main results are presented in Tables B = 0.041, q = 0.008). In addition, in females, increased thickness of the right postcentral sulcus , and increased volume of the right orbital  and postcentral gyri  was associated with more RRBI symptoms, while controlling for other autism symptoms. For males, RRBIs were associated with reduced volume of the right cerebellum cortex   and surface area  of the right intraparietal sulcus, and of thickness of the right orbital gyrus .Further, the relationship between RRBIs on the ADI-R and brain structure differed significantly for males and females on both thickness  and lateral orbital sulci  as well as right orbital gyrus , and increased surface area of the right supramarginal gyrus . In males, within-pair increases in current RRBIs, were associated with increased volume of the right pallidum . In addition, in males we observed associations when setting the threshold to q < 0.1, which included within-pair reduction of volume  and surface area  of the right postcentral sulcus . Finally, we tested the robustness of our findings in a sub-sample with very high data quality (n = 70). These analyses largely replicated our initial findings, but showed additional associations between cortical structure and RRBIs in females, in particular in the prefrontal cortex, but also a few associations between RRBIs and brain structure in males: the left lateral orbital sulcus (reduced surface area), ACC (increased thickness), and supramarginal gyrus (increased surface area and volume) .Table The present twin-study is the first to assess sex differences in the anatomy of brain networks associated with RRBI symptoms in autism. Significant associations were observed mostly within female pairs with largely varying frequencies and severities of RRBI symptoms and traits. In particular, the female twin with more RRBI symptoms had increased thickness of the right intraparietal sulcus. Additional alterations were found in the orbito-frontal areas, albeit without reaching statistical significance. Despite comparable within-pair differences in RRBIs, and comparable level of total autistic impairments, such associations with brains structure were not observed in males. Our results therefore suggest that, when controlling for many shared factors between twins, associations between RRBI symptoms and brain structure are mostly found in females, and involve in particular increased thickness of the cortex.Our observations correspond partly to the previous study on sex differences in the neuroanatomy of the motor networks in ASD , where bq < 0.1) in a region involved in executive function and decision making  [Thus, in addition to our study, these findings suggest mainly brain structural associations with RRBIs in females and not in males. Indeed, in our study there was a striking contrast between finding increased thickness in fronto-parietal regions in females, while there were hardly any associations between brain structure and RRBI’s in males. While Supekar and Menon report mostly primary motor regions, we observe sex-specific associations with RRBIs in females in a region involved in visuo-motor coordination and intention interpretation , that has also been shown to be involved in shifting attention and motor learning , in addial gyri) , 52. Theal gyri) . In addial gyri) , 52. It al gyri) –56. Thesal gyri) .The sex-specific findings could be an indication of etiological differences underlying symptom domains of ASD in males and females. Previously, interactions between sex and ASD diagnosis were observed for white matter connectivity density of the medial parietal lobe, of which the intraparietal sulcus is a part . HoweverOther than inherent etiological differences between males and females, an explanation of our sex-specific findings could be that the variance in brain structure within pairs was greater for females, therefore leading to significant associations in females but not males. Such increased brain structure differences, in combination with comparable differences in RRBI symptoms themselves, suggest greater cerebral and behavioral impairment in females for similar symptom levels. This observation might be a consequence of camouflaging. This entails that females need to have more severe RRBIs before even being noticed by their environment. Camouflaging leads to an underestimation of the true severity of autistic symptoms in females . In factAn additional alternative explanation might be that the observed reductions in volume in females are related to a more general and unspecific severity of autism symptomatology. However, re-running our analyses while regressing out highly correlated variance of other autism symptom domains and traits, thickness of both the intraparietal sulcus and orbital gyri were still associated with RRBI symptoms in females. Moreover, similar associations were observed when using RRBIs estimated with SRS-2. Compared to the SRS-2, assessing autistic-like traits in a short time frame (6 months), the ADI-R collects clinical RRBI symptoms and we used scores reflecting lifetime behaviors. Therefore, our patterns of RRBIs findings on the ADI-R and SRS-2 might indicate certain alterations in anatomy of the intraparietal sulcus and orbital gyri that are clinically relevant and robust for current or past presence of symptoms.Finally, future research is required to specifically assess which genetic and environmental factors contribute to neuroanatomical alterations in females with ASD and whether females are more sensitive to non-shared environmental factors compared to males. Non-shared environmental factors could in this case also entail the repetitive behaviors themselves, which, when present at an early age, reinforce preexisting structural alterations . Indeed,Taken together, our results point at the importance of investigating the female ASD phenotype, both on a behavioral and a neurobiological level, in order to understand the male and the female expression of the disorder. If future research is able to identify non-shared environmental factors that are differentially influencing the development of ASD in males and females, these could be targeted by interventions and give us more awareness of potential ASD risk factors for each sex.Although our study benefits from a unique sample of twins and a thorough clinical assessment and MRI analyses, some issues need to be addressed that warrant caution in interpreting the results. Although the total twin sample is large, the regression coefficient of the within-pair analysis is influenced only by the 37 pairs  that differed by at least one point on RRBIs, limiting the power of the sex-specific within-pair analysis. In addition, while the variability of symptoms on the ADI-R C and SRS-2 AM were adequate, the mean values of RRBIs in our sample were quite low, thus neuroanatomical differences might become more apparent in samples where the difference in RRBs is larger and participants exhibited more severe RRBs. However, our within-pair design enhances sensitivity for small associations that are otherwise masked by between pair variability and genetic variation. Indeed, when we re-ran our analyses across pairs, that is simply investigating the association between RRBIs and brain structure in this cohort, we found fewer significant results, showing that the within-pair design enhances our sensitivity to small differences. Hence, the within-pair analysis increases the ability to detect neural correlates of RRBs which might be subtler compared to the effects of age and shared genetic and environmental factors.In addition, the ADI-R assesses if RRBI’s have ever occurred in the participant’s lifetime, which means they do not necessarily need to be present now. However, as we replicate our results with the SRS-2 RRB scores, we believe that the ADI-R is an adequate measure of the impact of RRBI symptoms on brain structure.Further, our sample size did not permit to assess monozygotic twin pairs separately. The ratio between MZ/DZ was not exactly the same between males (50/38) and females (42/20), with the male group consisting of relatively more DZ twins, thus limiting our assessment of the impact of non-shared environmental factors in males. However, this difference was not statistically significant. Further, ADHD was twice as common among the males compared to females. Although this difference was not significant either, it needs to be taken into account because symptoms of ADHD such as inattention are probably related to RRBs . This woFurther, we allowed small segmentation errors in our data-set, due to the young age of the sample. Segmentation errors are known to be able to influence estimates of cortical thickness. Therefore, we might have missed significant associations due to noise. Re-running our analyses on a subset with high data quality replicated our initial findings, indicating that they were probably robust to quality issues. However, this replication generated additional regions showing increased cortical thickness in association with RRBIs in females. There were a few regions related to RRBIs in males as well. Thus, quality issues due to movement might have obscured some results in the main analyses, particularly in the males, as they were younger and younger participants moved more. Indeed, in the high-quality male subsample, we did see altered structure of the right supramarginal gyrus, left lateral orbital sulcus, and left anterior cingulate cortex. The overall pattern of results remained similar though, with increased thickness associated with RRBIs in particular in females.p value instead of a more stringent family-wise error (FEW) correction. However, replication in a larger sample is therefore needed to confirm our findings.Finally, our choice of ROIs is a compromise between reducing the number of comparisons and enhancing sensitivity for thus far unknown associations between RRBIs and brain structure, in particular in females. Thus, the number of ROIs chosen are relatively many since the brain correlates of RRBIs are not well established and we did not want to miss potentially meaningful association. At the same time, our choice of ROIs reduced the number of comparisons considerably compared to a whole-brain approach. Further, in order to not miss small but meaningful changes within pairs, we chose the rather lenient false discovery rate (FDR)-corrected In conclusion, this twin study shows that quantified features RRBI are mostly associated with brain anatomy alterations in females. The results add evidence to the hypothesis that there are etiological differences underlying ASD between males and females.Additional file 1: Figure S1. Within Pair distributions of ADI-R C scores. A within-pair difference per pair consists of the difference in score for RRBIs on ADI-R between 2 twins in a pair. The within-pair difference ranged from 0 to 5 points (B panel) in the cohort. Figure S2. The figure illustrates the within-pair difference model that was implemented in the analyses. The example shows within-pair difference associations in 3 twin pairs. Each line connects 2 individuals from one twin pair. In these examples, the individuals with a higher RRBI score on ADI-R compared to their co-twin, also had smaller right cerebellar cortex volumes. Figure S3. Within-pair association between RRBIs on ADI-R scores and thickness of the right intraparietal sulcus in males and females. Each dot represents one twin pair. For females (light-grey), there was a significant positive within-pair association between RRBIs on ADI-R and thickness of the right intraparietal sulcus.Additional file 2: Table S7A. Within-pair associations between cortical volume, surface area and thickness of neocortical regions of interest (ROIs) and RRB symptoms from SRS-2. Estimates (B), standard deviations (SD) and q-values (q) for the within pair associations between all surface-based neocortical measures  and RRBs as assessed by the SRS-2. A positive estimate indicates an increase in brain estimate related to more repetitive behavior symptoms. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). Table S7B. Within-pair associations between cortical volume of subcortical and cerebellar regions of interest (ROIs) and RRB symptoms from SRS-2. Estimates (B), standard deviations (SD) and q-values (q) for the within pair associations between subcortical & cerebellar volumes and RRBs as assessed by the SRS-2. A positive estimate indicates an increase in brain estimate related to more repetitive behavior symptoms. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. L. = Left, R. = Right. Table S8A. Within-pair model for RRBI’s (from ADI-R) predicting atomy of neocortical ROI’s, while controlling for social interaction deficits (from ADI-R). Estimates (B), standard deviations (SD) and q-values (q) for the linear associations between all surface-based neocortical measures  and RRBs as assessed by the ADI-R, while controlling for autistic symptoms from the social domain, as assessed by the ADI-R. A positive estimate indicates an increase in brain estimate related to more repetitive behavior symptoms. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. Table S8B. Within pair model for RRBI’s (from ADI-R) predicting atomy of subcortical and cerebellar ROI’s, while controlling for social interaction deficits (from ADI-R). Estimates (B), standard deviations (SD) and q-values (q) for the within-pair associations between subcortical measures (volumes only) and RRBs as assessed by the ADI-R, while controlling for autistic symptoms from the social domain, as assessed by the ADI-R. A positive estimate indicates an increase in brain estimate related to more repetitive behavior symptoms. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. L. = Left, R. = Right. Table S9A. Within-pair model for RRBI’s (from SRS) predicting atomy of neocortical ROI’s, while controlling for social cognition deficits (from SRS). Estimates (B), standard deviations (SD) and q-values (q) for the within pair associations between all surface-based neocortical measures  and RRBs as assessed by the SRS, while controlling for autistic symptoms from the social domain, as assessed by the SRS. A positive estimate indicates an increase in brain estimate related to more repetitive behavior symptoms. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. Table S9B. Within-pair model for RRBI’s (from SRS) predicting atomy of subcortical and cerebellar ROI’s, while controlling for social cognition deficits (from SRS). Estimates (B), standard deviations (SD) and q-values (q) for the within pair associations between subcortical measures (volumes only) and RRBs as assessed by the SRS, while controlling for autistic symptoms from the social domain, as assessed by the SRS. A positive estimate indicates an increase in brain estimate related to more repetitive behavior symptoms. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. L. = Left, R. = Right. Table S10A. Linear model assessing the interaction between age and RRBI symptoms (ADI-R) and neocortical regions of interest. Estimates (B), standard deviations (SD) and q-values (q) for the linear interaction associations between age (years) and RRBIs as assessed by the ADI-R on neocortical volume, surface area and thickness. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. Table S10B. Linear model assessing the interaction between age and RRBI symptoms (ADI-R) and subcortical regions of interest. Estimates (B), standard deviations (SD) and q-values (q) for the linear interaction associations between age (years) and RRBIs as assessed by the ADI-R on subcortical volumes. Bold text indicates significant associations  or associations with q-value < 0.10 (FDR-corrected). All results are FDR corrected. L. = Left, R. = Right.


Introduction: The coordination of swallowing and respiration is important for safety swallowing without aspiration. This coordination was affected in Parkinson disease (PD). A noninvasive assessment tool was used to investigate the effect of an easy-to-perform and device-free home-based orolingual exercise (OLE) program on swallowing and respiration coordination in patients with early-stage PD.Materials and Methods: This study had a quasi-experimental before-and-after exercise program design. Twenty six patients with early-stage PD who were aged 62.12 ± 8.52 years completed a 12-week home-based OLE program. A noninvasive assessment tool was used to evaluate swallowing and respiration. For each patient, we recorded and analyzed 15 swallows  before and after the home-based OLE program. Oropharyngeal swallowing and its coordination with respiration were the outcome measures. The frequency of piecemeal deglutition, pre- and post-swallowing respiratory phase patterns, and parameters of oropharyngeal swallowing and respiratory signals  were examined.Results: The rate of piecemeal deglutition decreased significantly when swallowing 10- and 20-mL water boluses after the program. In the 1-mL water bolus swallowing trial, the rate of protective pre- and post-swallowing respiratory phase patterns was significantly higher after the program. For the parameters of oropharyngeal swallowing and respiratory signals, only the amplitude of laryngeal excursion was significantly lower after the program. Moreover, the volume of the water bolus significantly affected the SRP and duration of submental sEMG when patients swallowed three small water bolus volumes .Conclusion: The home-based OLE program improved swallowing and its coordination with respiration in patients with early-stage PD, as revealed using a noninvasive method. This OLE program can serve as a home-based program to improve swallowing and respiration coordination in patients with early-stage PD. The incidence of oropharyngeal dysphagia (OD) was reported to be high in patients with Parkinson disease (PD) . SubclinRehabilitation swallowing therapy (RST) includes indirect and direct swallowing training. Indirect swallowing training without oral feeding is safer than direct swallowing training involving oral feeding, which requires supervision under a therapist in the beginning. Indirect swallowing training includes thermal stimulation, oral motor exercises, and lingual exercises. Exercises are focused on strengthening the tongue muscles, submental muscles, and suprahyoid muscles. Isometric tongue-strengthening exercises have been reported to improve swallowing function in elderly people and patients with stroke, and performing a sensorimotor task has been suggested for strengthening specific muscles , 7. HoweTongue movements are essential for safe swallowing and effective swallowing and respiration coordination. Tongue movement is controlled by the hypoglossal nerve, and the hypoglossal nucleus is located in the caudal pons and ventral medulla , which aPrevious studies have reported swallowing and respiratory dysfunction in PD –25, but Ethics approval was granted by the Institutional Review Board of our hospital (no. 102-5410B). Each participant was able to understand verbal instructions and signed informed consent prior to participation.In the home-based OLE program, we recruited 26 patients with PD who were referred from the department of neurology. All of these patients completed the training program and follow-up noninvasive swallowing and respiration examinations after undergoing 12 weeks of the exercise program. The enrolled patients had stage I–III PD according to the Hoehn and Yahr disability scale Table . All patThe easy-to-perform and device-free home-based OLE program was designed with each cycle including two repetitions of effortful dry  swallowing, two repetitions of tongue protruding out Figure , followeSimilar to our previous studies –28, we uNoninvasive assessment of swallowing and respiration coordination was conducted at two time points: at the baseline (before the program) and after the program.1. Piecemeal deglutition : Multiple swallows are observed when the bolus volume fed in the mouth exceeds the dysphagia limit.2. Pre- and post-swallowing respiratory phase patterns : The expiration–expiration (Ex–Ex) pre- and post-swallowing respiratory phase pattern is the major and protective swallowing respiratory phase pattern.3. Parameters  of oropharyngeal swallowing and respiratory signals: These parameters included swallowing respiratory pause (SRP), onset latency (OL), total excursion time (TET), excursion time (ET), second deflexion of laryngeal excursion, amplitude and duration of submental sEMG activity, and amplitude of laryngeal excursion.All the aforementioned parameters have been defined and used to analyze swallowing and its coordination with respiration in our previous studies –28.P-value < 0.05 was considered statistically significant. Using G*Power 3, we calculated a sample size of more than 27 for repeated measures ANOVA to detect large effects (0.27)  was used for all statistical analyses. For each patient, three swallowing trials of each bolus volume were conducted, with a total of 15 swallows. The chi-square test or Fisher exact test was used to analyze patient data on piecemeal deglutition (10 and 20 mL) and pre- and post-swallowing respiratory phase patterns , which are expressed as numbers and frequencies. Oropharyngeal and respiratory signals alone  were averaged and analyzed. Repeated-measures analysis of variance was used to examine the interaction and main effect. Independent variables were SRP and oropharyngeal parameters, and dependent variables were measurements obtained before and after exercises and three small bolus volumes . The level of α was set at 0.05. A s (0.27) , with 80n = 26).Tables P < 0.001 and P = 0.001, respectively; Table Compared with the baseline, the probability of piecemeal deglutition decreased significantly after the program in patients with PD in both 10- and 20-mL water bolus swallowing trials  respiratory phase patterns tended to increase after the program in swallowing trials of the five water bolus volumes without piecemeal deglutition. However, a significant difference was observed before and after the program in only the 1-mL water bolus swallowing trial without piecemeal deglutition , indicating that SRP was not longer after the program in patients with PD. However, SPR significantly differed among the different water bolus volumes  and among the three small bolus volumes  and among the three small bolus volumes , and the ET did not differ significantly among the three small bolus volumes . Moreover, no significant difference was observed in the second deflexion among the three small bolus volumes . The water bolus volume significantly affected the parameters when patients swallowed the three small bolus volumes  as well as among the three small bolus volumes , but it did not differ significantly among the three small bolus volumes , a high rate of protective respiratory phase patterns, and a low amplitude of laryngeal excursion were observed after the 12-week home-based OLE program. Regarding the temporal parameters of oropharyngeal swallowing and respiratory signals, the duration of the SRP, TET, ET, and second deflexion associated with swallowing the three small water bolus volumes  tended to be shorter after the OLE program than before the program in patients with PD; however, this difference was not statistically significant. Taken together, our data derived from the noninvasive swallowing and respiration assessment indicate that the coordination of swallowing and respiration in patients with early-stage PD improved after the 12-week home-based OLE program.According to our review of the literature, this is the first study to evaluate the effects of an easy-to-perform and device-free home-based OLE program on swallowing and respiration coordination by using a noninvasive examination. Our data demonstrate this home-based OLE program including effortful dry  swallowing and lingual exercises exerted a positive effect on swallowing and respiration coordination in patients with early-stage PD.Swallowing is a consequence of movement patterns. Accordingly, task-specific exercises for swallowing training are based on the effectiveness of skill training in the limbs –34. TaskPatients with PD have poor oral control  and a hiFor clinical application, this home-based OLE program may be useful in early-stage PD and can be used as a regular home exercise program. An early intervention to prevent the deterioration of oral motor and swallowing function is important. Designing an easy, simple, and device-free home-based program is the most suitable option. Future studies with similar designed investigations and programs will be important for patients with later stages of PD. The therapeutic effects of other oral motor training programs and respiratory muscle-strengthening exercises are also crucial in early PD.Our study has some limitations. First, this study did not apply a randomized design. Because the variable clinical symptoms and signs and heterogeneous courses in PD, performing a randomized study is difficult. However, PD is a chronic progressive disease, and spontaneous recovery is not expected with time. Second, our patients were in the stationary status receiving an unaltered protocol of anti-PD medication for more than 2 months and were regularly followed up at the outpatient clinics of the neurological department. Thus, our conclusion may not be generalizable. Third, we did not recruit patients with late-stage PD or those with severe dysphagia requiring tube feeding; thus, the results of this study cannot be generalized to such patients. Finally, no long-term follow-up was conducted after the training program in our study; thus, the long-standing effects of the training program could not be investigated.The easy-to-perform and device-free home-based OLE program exerted a positive effect on oropharyngeal swallowing and respiration coordination in patients with early-stage PD. This OLE program can be useful in clinical application for dysphagia care in early PD.C-MW, Y-WH, W-YS, and C-SH performed the research. C-MW and Y-RW designed the research study. C-MW and W-YS contributed essential reagents or tools. C-MW and W-YS analyzed the data. C-MW and Y-RW wrote the paper.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.


Cyclic and linear nucleotides are key elements of the signal transduction networks linking perception of the environment to specific cellular behavior of prokaryotes. These molecular mechanisms are particularly important in bacteria exposed to different, and frequently simultaneous, types of extreme conditions. This is the case in acidithiobacilli, a group of extremophilic bacteria thriving in highly acidic biotopes, that must also cope with significant variations in temperature, osmotic potentials and concentrations of various transition metals and metalloids. Environmental cues sensed by bacteria are transduced into differential levels of nucleotides acting as intracellular second messengers, promoting the activation or inhibition of target components and eliciting different output phenotypes. Cyclic (c) di-GMP, one of the most common bacterial second messengers, plays a key role in lifestyle changes in many bacteria, including acidithiobacilli. The presence of functional c-di-GMP-dependent signal transduction pathways in representative strains of the best-known linages of this species complex has been reported. However, a comprehensive panorama of the c-di-GMP modulated networks, the cognate input signals and output responses, are still missing for this group of extremophiles. Moreover, little fundamental understanding has been gathered for other nucleotides acting as second messengers. Taking advantage of the increasing number of sequenced genomes of the taxon, here we address the challenge of disentangling the nucleotide-driven signal transduction pathways in this group of polyextremophiles using comparative genomic tools and strategies. Results indicate that the acidithiobacilli possess all the genetic elements required to establish functional transduction pathways based in three different nucleotide-second messengers: (p)ppGpp, cyclic AMP (cAMP), and c-di-GMP. The elements related with the metabolism and transduction of (p)ppGpp and cAMP appear highly conserved, integrating signals related with nutrient starvation and polyphosphate metabolism, respectively. In contrast, c-di-GMP networks appear diverse and complex, differing both at the species and strain levels. Molecular elements of c-di-GMP metabolism and transduction were mostly found scattered along the flexible genome of the acidithiobacilli, allowing the identification of probable control modules that could be critical for substrate colonization, biofilm development and intercellular interactions. These may ultimately convey increased endurance to environmental stress and increased potential for gene sharing and adaptation to changing conditions. Acidithiobacillus are chemolithoautotrophic Gram-negative bacteria that thrive in environments with few nutrients, high concentrations of heavy metals and extreme acidity. They obtain energy from the oxidation of reduced inorganic sulfur compounds (RISCs), including many metal sulfides, one of the most abundant mineral classes on earth ; the presence/absence of flagella-based motility  , cyclic E. coli. In this neutrophilic bacterium, amino acid deficit promotes the “stringent response,” a series of physiological changes driven by an increase of intracellular levels of (p)ppGpp. Acting at transcriptional and post-traductional levels, (p)ppGpp regulates biosynthesis of amino acid and ribosomal proteins  and (p)ppGpp have been studied for decades in the context of nutrient deprivation in proteins  and reprproteins . Furthergulation . More rearvation . Cyclic-arvation , althougarvation .A. ferrooxidans , was analyzed. Finally, to evaluate the interconnection of predicted control/effector/target gene modules, the sub-cellular localization of c-di-GMP related proteins and the concurrence of signal-sensing associated domains were examined.Taking advantage of the increasing number of sequenced genomes of the taxon, here we address the challenge of disentangling the nucleotide-driven signal transduction pathways in this group of polyextremophiles by using a comparative genomics strategy. First, the presence, occurrence, and conservation of nucleotide second messenger related gene products in available bacterial genomes of Acidithiobacillus spp. and Thermithiobacillus tepidarius DSM 3134 (outgroup) were downloaded from the NCBI repository as of June 2018 and filtered based on quality and completion criteria (Complete and draft genomes sequences (chromosomes and plasmids) for criteria . All 35 criteria . Completcriteria , COG  with Usearch v1.2.22 . ProteinThe FastTree program v2.1.7  was used1.All candidate proteins were analyzed with additional resources, including SignalP v4.0  and PhobComplete genomes  were used for identification of putative MGEs using the following programs: AlienHunter , PAI-DA Protein and genome comparisons were performed with Blastp and Blastn respectively and visualized with Artemis Comparison Tool (ACT) v1.0 . FiguresAcidithiobacillus species  and 16 plasmids] belonging to  species , compris species . WorkfloAcidithiobacillus genome analyzed. Surprisingly, hydrolases for the degradation of c-di-AMP dinucleotide were also identified in all genomes, even if the synthesizing diadenylate cyclases (DACs) appeared to be absent. Globally, these results suggest that signaling nucleotides are relevant as second messenger molecules for bacteria thriving in acidic econiches, as they are in other environments. Each of the pathways uncovered is analyzed in further detail in the sections below and, when possible, the implications in acidithiobacilli ecophysiology are inferred.Synthesis functions were identified in all 35 chromosomes for every signaling nucleotide targeted, except c-di-AMP . All genAcidithiobacillus spp. Without exception, every genome analyzed was found to encode a single copy of the large (p)ppGpp synthetases belonging to the RSH protein superfamily (RelA/SpoT Homolog), named after the discovery of RelA and SpoT proteins in Escherichia coli (Aci onwards (see below).We first assessed the presence, occurrence, and conservation of guanosine tetra and/or pentaphosphate synthesis and degradation functions in hia coli . The acirelA and spoT), arisen through a gene duplication event of an ancestral bifunctional “Rel” protein (Acidithiobacillus genus only possess one long RSH (spoT) per genome. This is consistent with the recent re-classification of this acidophilic taxon as a new class (Acidithiobacillia), ancestral to both the γ- and β-proteobacteria , an ortholog of the gppA gene encoding for the pppGpp phosphohydrolase ppGpp hydrolysis  group from ATP to the 3′ position of GDP and GTP, respectively. SpoTN domain  instead er ppGpp . Despiteydrolase . This isydrolase . All Spodrolysis , and areAci accessory domains are TGS , ZFD (zinc-finger domain), and RRM (RNA recognition motif) (HYD-SYN-TGS-ZFD-RRM configuration), which are widely conserved in the protein family. The presence and conservation of the TGS domain in SpoTAci suggests that its synthetase activity may be tiriggered by amino acid and/or fatty acid starvation (Acidithiobacillia genomes screened (data not shown).In well-studied microbial models, synthesis of (p)ppGpp is activated by different environmental stresses, including temperature changes e.g.,  and limiarvation . Congruearvation , most ofAcidithiobacillia long RSH protein encoding genes occur in a highly conserved gene cluster, which resembles the spoT operon of E. coli both in structure and sequence (spoTAci are gmk and rpoZ. One of the best known (p)ppGpp mediated effects is its global control of gene expression, through direct binding to the RNA polymerase omega subunit (RpoZ). In this way (p)ppGpp simultaneously inhibits rRNA synthesis and the expression of ribosomal proteins ppGpp binding  kinase, is a key player in de novo pathway for GTP synthesis catalyzing the formation of GDP from GMP (plus ATP)  required for (p)ppGpp binding (gmk and AcispoT in the same operon and amino acidic sequence level conservation of GMK (data not shown) suggests that in Acidithiobacillia GMK is also inhibited by (p)ppGpp. The decrease of intracellular GTP levels may have an impact on the synthesis of others nucleotide messengers based in GTP such as c-di-GMP.sequence . Conservproteins , and actproteins . In the  binding  (Supplem binding . In turnlus ATP) . In othelus ATP) . The gmk binding  (Supplem binding . The co-A. thiooxidans (6 out of 10) and A. ferrivorans (2 out of 7) one copy of Small Alarmone Hydrolases (SAHs) containing just the HYD domain (In some strains of D domain  were ideD domain . PresentD domain .Acidithiobacillia corresponds to the one driven by cyclic AMP (cAMP). cAMP is synthesized from ATP by cognate nucleotidyl cyclases  were detected in any of the species of the genus.A second highly conserved signal transduction pathway in cyclases . ACs arecyclases . Every AAeromonas (Ae.) hydrophila and Yersinia pestis (YpAC-IV), which are formed by a single CYTH domain (pfam01928) and exhibit thermophilic properties (Aci)  performs optimally at 37–45°C and pH 8.5, suggesting that cAMP is synthetized under different in vivo situations by either one of the enzymes  . In Ae.  enzymes . DespiteAcidithiobacillus ACAci, a “conserved histidine alpha-helical domain” (CHAD) is present at the proteins C-terminus (phosin given its capacity to bind polyphosphate (polyP) granules , catalyzing the reversible conversion of the terminal phosphate of ATP into polyP  has been proposed to work as phosphoesterase through a two-metal reaction mechanism, with the AC activity being a secondary reaction . This gene encodes a dual function enzyme  central to the folate (vitamin B9)-dependent C1 metabolism, highly conserved in all domains of life for its role in the biosynthesis of key biological building blocks   . In thisAcidithiobacillus genomes analyzed encode for cAMP-receptor proteins (CRPs), with the typical domain architecture described for E. coli ; 3 Acidithiobacilllus sp. SH; 5 T. tepidarius; 3-5 A. caldus; 4-6 A. ferrivorans, 4-7 A. thiooxidans; 2-7 A. ferrooxidans; 9 A. albertensis], being A. caldus and T. tepidarius CRPs the most divergent. Intriguingly, amino acidic sequence alignment of the Acidithiobacillia CRP orthologs showed that the residues involved in cAMP binding  . All Aci E. coli , i.e., a binding  are not  binding ] and sub binding , howeverAcidithiobacillia genomes. This cyclic nucleotide is best known for its role as second messenger controlling the transition from motile planktonic state to the attached multicellular state achieved in biofilms  dependent signaling in the targeted biofilms . Within Acidithiobacillus species analyzed encode in their genomes a vast diversity of genes linked to c-di-GMP metabolism , including opposing diguanylate cyclase (DGCs) and phosphodiesterase (PDEs) activities  to be secreted through outer membrane by the type 2 secretion system (T2SS) protein complex  rather than degradation related proteins (2% in EAL-only and 0% in HD-GYP) . Twenty  complex . Two subAcidithiobacillia are shown in Period circadian protein, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded protein), GAF , REC (response regulator receiver domain), Protoglobin [or globin coupled sensor (GCS)], and CZB (Chemoreceptor Zn-Binding domain). Some of these domains are very well-characterized and thus their role in signal transduction can be robustly inferred. For example, the REC domain is typically present in the receiver element (or response regulator) of two-component signal transduction systems  and nearly half of the GGDEF-only proteins (42%) contain partner domains . Most co systems  and unde systems . Both A.ruginosa . On the ruginosa . Just a F domain . Recentlc-di-GMP . Thus, tc-di-GMP . Using dc-di-GMP , O2   , which huration) . On the Acidithiobacillus species . The highest net number of DGCs/PDEs observed occurred in A. thiooxidans GD1-3, with a total of 34 c-di-GMP metabolism elements predicted. In turn, the lowest number occurred in A. ferrooxidans strains. The type of strains of the A. ferrooxidans, ATCC 23270T and strain ATCC 53993, had only four predicted c-di-GMP metabolism proteins, the lowest number in the whole Acidithiobacillia class, potentially representing the minimal essential set of DGCs/PDEs for the taxon. Yet, only one of them is conserved through the Acidithiobacillia class members , and two other across A. ferrivorans strains . The fourth gene  is shared only with A. thiooxidans. This distribution suggests that DGCs/PDEs shared with specific groups may have been gained in informative ecological contexts  that may provide clues on the specific effectors targeted by these and/or other specific control proteins.The abundance and diversity of c-di-GMP metabolism enzymes is thought to reflect the extent and complexity of the c-di-GMP dependent network in any given microorganism. In this respect, we observed important differences between  species , in bothA. ferrooxidans, one of the more versatile representatives of the taxon xSxxG , which are homologs to bacterial cellulose synthase A subunit (BcsA), and therefore may synthetize cellulose or some other glucose-based exopolysacharrides needed for attachment and extracellular matrix construction. In Komagataeibacter xylinus, cellulose synthesis depends on the presence of the cellulose synthase complex  involved in the transcriptional regulation of flagella and EPS production. In P. aeruginosa, the binding of c-di-GMP in the AAA domain of FleQ determines a slight decrease in the transcription of flagellar genes, but simultaneously causes a great increase in the transcription of the pelABCDEFG operon required for the synthesis of PEL exopolysaccharide and the formation of biofilms  are likely to have co-evolved to accomplish a common function, we decided to inspect this aspect in detail as a strategy to uncover potential c-di-GMP responsive control modules. Using genome information and comparative genomics tools, we mapped c-di-GMP related elements  to the core and flexible/mobile genome of five completely sequenced A. ferrooxidans ATCC 23270  was identified in the A. caldus ATCC 51756T chromosome (and conserved in a SM1), which contains five predicted c-di-GMP elements . One of the GGDEF proteins encoded by genes present in this c-di-GMP “hot spot” , Aca1319 (formerly Aca1413), has already been experimentally characterized , being one of the few Acidithiobacillus mutants obtained world-wide  do possess all genes required to construct a functional c-di-AMP signaling pathway, including synthesis, degradation and effectors proteins (data not shown). Therefore, evidence gathered in this work suggests that extracellular signal transduction pathways relying on cyclic nucleotidic second messengers may indeed exist in acidic econiches and raises the intriguing possibility that these nucleotides may mediate both intra- and inter-species communication. The existence and role of these interaction merits further exploration.Bioinformatic analysis performed in this study failed to identify proteins carrying the domains required for c-di-AMP synthesis ppGpp, cAMP, and c-di-GMP. As in other bacteria, in Acidithiobacillus the (p)ppGpp synthesis through SpoTACI is predicted to respond to nutrient limitation, reprograming global gene expression through direct binding to RpoZ RNA polymerase subunit. Cyclic AMP nucleotide synthesis in the acidithiobacilli seems to be limited by ATP availability, yet functional association-based inference suggests it could also be regulated by the presence of polyP. To our knowledge, this link has not been pointed out nor explored previously in the acidithiobacilli. The most versatile and diverse signaling pathway uncovered in the Acidithiobacillia was the one relying on c-di-GMP. On the basis of diverse partner sensor, synthesis and degradation domains, this pathway is predicted to transduce environmental cues  as well as intracellular signals (such as phosphorylation cascades), into differential levels of intracellular c-di-GMP. These, in turn, are predicted to regulate flagellar based motility, substrate attachment, and subsequent biofilm development through binding to diverse effector proteins included PilZ domain, FleQ and PelD proteins encoded in the gene vicinity of the DGCs and/or PDEs or confined to specific MGEs. All the (p)ppGpp, cAMP, and c-di-GMP second messenger systems, may cross-talk with each other through different components of the respective pathways (This study is the first comprehensive genomic analysis of nucleotide second messengers-based signaling pathways in polyextremophilic bacteria belonging to the pathways . Accordihttps://figshare.com/s/63b7234a67db25ff7a8b; 10.6084/m9.figshare.7553921.Protein alignments of MC and RQ conceived and supervised the study and metabolic reconstruction analysis. AM-B and CR-V carried out the sequence processing and bioinformatic analyses. NG and MD contributed in data analysis. MC, AM-B, and RQ analyzed and interpreted the data and wrote the manucript. All authors read 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.


Currently, there are three main surgical approaches for resectable esophageal cancer: open transthoracic esophagectomy (OTE), conventional minimally invasive esophagectomy (MIE) and robot-assisted esophagectomy (RAE). Previous studies had demonstrated the better short-term outcomes in MIE or RAE when compared to OTE, respectively. However, to date, no prospective study was designed to compare these two minimally invasive approaches (MIE and RAE). The primary objective of this study is to compare the outcomes on survival, safety and efficacy, quality of life between RAE and MIE in the treatment for resectable esophageal squamous cell carcinoma (ESCC).n = 180) or MIE (n = 180) group. The follow-up visits will be scheduled at 3, 6, 9, and 12 months in the first two years, and then every 6 months until the end of the study. During the follow-up period, clinical data and quality of life questionnaires will be examined. The primary endpoint is the 5-year overall survival (OS). The secondary endpoints are 3-year OS, 5-year disease-free survival (DFS), short-term outcomes as well as quality of life.This study is designed as a multicenter, prospective, randomized, non-inferiority phase III clinical trial, investigating the safety and efficacy of RAE compared with MIE in the treatment of resectable ESCC. Eligible patients are randomly assigned to either RAE . The trial was prospectively registered.ClinicalTrial.gov:  Esophageal cancer is the seventh common malignant tumor and ranks sixth in tumor-related mortality worldwide in 2018. In terms of the histological subtypes, adenocarcinoma is frequently observed in Europe and the United States, while squamous cell carcinoma is the predominant form of Asian populations . The primary endpoint of our controlled trial is 5-year overall survival. According to our hypothesis, RAE will result in at least similar oncological outcomes and long-term quality of life compared with MIE.Although RAE is reported to improve the efficiency of lymph node dissection, it is unclear whether more extensive lymph node dissection could result in a significant survival advantage. Fewer studies were designed to focus on comparing long-term survival between RAE and MIE. Park et al. reported that the 5-year overall survivals were not different between the two groups (69% in RAE vs. 59% in MIE,


Adenosine-to-inosine RNA editing can markedly diversify the transcriptome, leading to a variety of critical molecular and biological processes in mammals. Over the past several years, researchers have developed several new pipelines and software packages to identify RNA editing sites with a focus on downstream statistical analysis and functional interpretation.Here, we developed a user-friendly public webserver named MIRIA that integrates statistics and visualization techniques to facilitate the comprehensive analysis of RNA editing sites data identified by the pipelines and software packages. MIRIA is unique in that provides several analytical functions, including RNA editing type statistics, genomic feature annotations, editing level statistics, genome-wide distribution of RNA editing sites, tissue-specific analysis and conservation analysis. We collected high-throughput RNA sequencing (RNA-seq) data from eight tissues across seven species as the experimental data for MIRIA and constructed an example result page.https://mammal.deepomics.org.MIRIA provides both visualization and analysis of mammal RNA editing data for experimental biologists who are interested in revealing the functions of RNA editing sites. MIRIA is freely available at  RNA editing is defined as a critical post-transcriptional regulatory RNA-processing event (excluding RNA splicing) that generates an RNA transcript with a primary nucleotide sequence different from its gene. In mammals, the most common form of RNA editing, A-to-I RNA editing, is catalysed by the ADAR family of enzymes (Adenosine Deaminase that Acts on RNA) , 2, and Recently, increasing number of mammalian RNA editing databases have been published –22, therMIRIA is a new tool that was designed for RNA editing analysis in mammals. Users need to provide a compressed file (.zip) containing all RNA editing files from different mammals. The architecture of the zip file has two layers. The first layer contains all species folders , and the second layer, which is inside each species folder, contains all RNA editing files of the species . The format of the single RNA editing file (.res) is listed below (Table Adenosine-to-inosine (A-to-I) editing is the most common form of editing in mammals. As such, the percentage of A-to-G editing, an indicator of A-to-I editing, is an important measurement to indirectly assess the detection accuracy of the RNA editing sites. Before calculating the percentage of A-to-G editing, we classified all editing sites into one of three categories, namely, those in Alu regions, those in repetitive non-Alu regions and those in non-repetitive regions. We performed the repeat region annotation using the RepeatMasker file downloaded from the UCSC Table Browser  . The peMIRIA can annotate RNA editing sites with a variety of useful genomic features. First, we used SnpEff , a genomTo examine the editing level statistics of each tissue in the uploaded data, we determined the editing level of each RNA editing site as the ratio of the number of reads supporting this site to the number of reads covering this site. The overall editing level statistics of each tissue were displayed using a boxplot Fig. e.For the statistical analysis of RNA editing sites on a genome-wide level, each chromosome was partitioned into contiguous 1-Mb windows, and the total number of RNA editing sites was calculated within each window. Thereafter, an interactive Circos graph was generated to compare the different RNA editing numbers between the different tissues on a genome-wide level  from the overview page of the job results interface.The MIRIA website was built using the Django Python Web framework  coupled https://mammal.deepomics.org/demo/.To evaluate the MIRIA webserver, we collected 55 RNA-seq datasets from seven mammals  as the test data. For each mammal, seven or eight tissue samples  were used. The RNA-seq data were downloaded from the National Center for Biotechnology Information (NCBI) SRA database. The genomes and annotation files of the mammals were downloaded from the ENSEMBL database . RepeatMWe developed the MIRIA to provide both visualization and analysis of mammal RNA editing data. MIRIA enables experimental biologists without any computational programming skills to perform a diverse range of analyses including RNA editing type statistics, genomic feature annotations, editing level statistics, genome-wide distribution of RNA editing sites, tissue-specific analysis and conservation analysis. For every analysis, the result is presented with a visualized graph and can be downloaded as a publication-ready format. In general, with the functions of the MIRIA designed for mammal RNA editing data, we believe that this webserver will be a valuable resource for experimental biologists who are interested in revealing the functions of RNA editing sites.Project name: MIRIAProject home page:https://mammal.deepomics.org/Operating system(s): Platform independentProgramming language: PythonOther requirements: Chrome, Safari, Firefox or IELicense: GNU GPLAny restrictions to use by non-academics: NoneAdditional file 1: Table S1. The data source from NCBI.


Clinicopathological characteristics alone are not enough to predict the survival of patients with cervical squamous cell carcinoma (CESC) due to clinical heterogeneity. In recent years, many genes and non-coding RNAs have been shown to be oncogenes or tumor-suppressors in CESC cells. This study aimed to develop a comprehensive transcriptomic signature for CESC patient prognosis. Univariate, multivariate, and Least Absolute Shrinkage and Selection Operator penalized Cox regression were used to identify prognostic signatures for CESC patients from transcriptomic data of The Cancer Genome Atlas. A normalized prognostic index (NPI) was formulated as a synthetical index for CESC prognosis. Time-dependent receiver operating characteristic curve analysis was used to compare prognostic signatures. A prognostic transcriptomic signature was identified, including 1 microRNA, 1 long non-coding RNA, and 6 messenger RNAs. Decreased survival was associated with CESC patients being in the high-risk group stratified by NPI. The NPI was an independent predictor for CESC patient prognosis and it outperformed the known clinicopathological characteristics, microRNA-only signature, gene-only signature, and previously identified microRNA and gene signatures. Function and pathway enrichment analysis revealed that the identified prognostic RNAs were mainly involved in angiogenesis. In conclusion, we proposed a transcriptomic signature for CESC prognosis and it may be useful for effective clinical risk management of CESC patients. Moreover, RNAs in the transcriptomic signature provided clues for downstream experimental validation and mechanism exploration. Cervical cancer (CC) is still the fourth most common cancer in women . DespiteBy comprehensively characterizing various molecules  in 100s of CC samples, The Cancer Genome Atlas (TCGA) has provided a comprehensive way to understand CC The . EnormouHowever, some limitations should be noticed: (1) Previous studies focused on single omics independently, and there lacks a whole transcriptomic analysis which may provide more comprehensive and robust discovery . (2) ProTo address these limitations, we submit the transcriptomic data of CESC patients to a Least Absolute Shrinkage and Selection Operator (LASSO) penalized MCA  to ident1 repository in January 2018. Search strategies can be obtained in Section I of the Level 1 clinical data, level 3 transcriptomic sequencing data, and the corresponding metadata of CCs were retrieved and downloaded from TCGAClinical eXtemsible Markup Language files were parsed by R “XML” package and the R code can be achieved in Section II of the Kaplan–Meier (KM) survival analysis with log-rank test was applied to evaluate the prognostic effects of age at diagnosis, clinical stage, menopause status, ethnicity, birth control pill usage, tobacco usage, and lymphovascular invasion for CESC. Furthermore, MCA with demographic and clinicopathological characteristics as covariates was adopted to evaluate their independence for CESC prognosis.The miRNA isoform sequencing data only include miRNAs while the gene sequencing data include both messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs). For convenience of description, we termed mRNA and lncRNA as gene and further termed miRNA, mRNA and lncRNA as RNA.Associations between OS and RNA expression profiles were preliminarily evaluated by univariate Cox regression analysis (UCA). The proportional hazards (PH) assumption was tested by Schoenfeld residual  and a unFor miRNAs, stepwise MCA was applied to preliminarily survival associated miRNAs to construct an independent miRNA-only expression signature for CESC prognosis. The Bayesian information criterion (BIC)  was adopBecause the number of preliminarily survival associated genes was comparable to the number of samples, a LASSO penalized MCA with 10-fold cross validation and 1000 iterations was adopted to select genes by penalizing low regression coefficients exactly to zero. To alleviate the local minimum problem, we repeated the LASSO penalized MCA 10 times with different initializations and the model that achieved the minimal partial likelihood was adopted. Furthermore, stepwise MCA was applied to genes selected by the LASSO penalized MCA to construct an independent gene-only expression signature for CESC prognosis.Finally, a transcriptomic signature for CESC prognosis was constructed by stepwise MCA with gene-only signature and miRNA-only signature as covariates.A normalized prognosis index (NPI) defined as the standard form of a linear combination of the observed values weighted by the regression coefficients was adopted as a synthetical index for CESC prognosis. Specifically,jth element of PI is the prognostic index of the jth patient, i.e.where PI is a prognostic index vector and the i is the regression coefficient of the ith variable ; ijG is the observed value of the ith variable in the jth sample . For miRNA-only signature, gene-only signature, the integrated RNA signature , and the previously identified prognostic signatures, we termed the corresponding NPI as miRNA-NPI, gene-NPI, RNA-NPI, and pre-NPI, respectively.βCESC patients were stratified into a high-risk group (NPI > 0) or a low-risk group (NPI < 0) based on NPI. OS between the high-risk group and the low-risk group was compared by KM survival analysis. MCA was used to evaluate the independence of various NPIs and clinical factors. The abilities of various NPIs to predict CESC patient survival outcome were assessed and compared by calculating the area under the curve (AUC) of the time-dependent receiver operating characteristic (ROC) at 3, 5, and 10 years, respectively.2 was adopted for gene ontology and pathway enrichment of the prognostic mRNAs and targets of the prognostic miRNA.Unlike other mRNA target prediction software just based on sequence alignment, miRTarBase provided experimentally validated mRNA targets of miRNA. Both strongly and weakly validated mRNA targets of the prognostic miRNA were obtained from miRTarBase (version 7.0) . MetascaP-value less than 0.05 or adjusted P-value less than 0.1 was considered to be significant. All analyses were performed by R software. Non-parametric survival analysis, semi-parametric survival analysis, and PH assumption test were performed by survival and survminer packages. LASSO penalized MCA was conducted by textitglmnet package. Multiple test correction was conducted by fdrtool package. Time-dependent ROC analysis was conducted by timeROC package.P-values of 0.598 and 0.947  and 309 samples for gene sequencing . Due to small number of metastatic and normal CC samples, we only analyzed the primary CC samples. Based on the inclusion criteria and low-expressed RNA filtering , 214 CES Figures , respectn = 106). Considering age at initial diagnosis, clinical stage, and tobacco usage as covariates , MCA revealed that only clinical stage was an independent prognostic clinicopathological characteristic . Demographic and clinicopathological characteristics for TCGA-CESC cohort are summarized in Table Univariate Cox regression analysis (UCA) after FDR correction , the gene-NPI , and the RNA-NPI  were significant predictors for OS of CESC patients revealed by UCA. However, MCA revealed that neither miRNA-NPI  and six mRNAs . It is natural to wonder if there are any mRNA targets of the non-coding RNAs in the transcriptomic signature. Interestingly, CXCL2 was reported to be a direct target of hsa-miR-532-5p in hepatocellular carcinoma and this miRNA-gene interaction inhibited hepatocellular carcinoma cell proliferation and metastasis . HoweverFor the protective RNAs in the transcriptomic signature, hsa-miR-532-5p was shown to be involved in many cancers either as a tumor suppressor or an oncogenic-miRNA . HoweverAmong the risky RNAs in the transcriptomic signature, Tyrosylprotein sulfotransferase 1 (TPST1) is an enzyme responsible for catalysis of tyrosine sulfation. Previous studies revealed that TPST1 could sulfate the tyrosine of C-X-C motif chemokine receptor (CXCR4)  and the P-value in multiple testing. (2) The transcriptomic signature was identified based on TCGA data-mining and further independent validations and mechanism explorations are in need. (3) Complete mechanisms could not be revealed by the transcriptomic signature itself. However, the transcriptomic signature may be of potential applications for clinical management of CESC patients. Specifically, we can measure the expressions of the transcriptomic signature in CESC patients and calculate the NPIs for the patients based on the measured expression values. Furthermore, CESC patients can be stratified as high-risk and low-risk based on their NPIs. Finally, for high-risk patients, more aggressive therapies such as high-dose chemoradiotherapy may be given. Moreover, hsa-miR-532-5p, RBM38, EGLN1, and DLEU1 were demonstrated as both oncogenes and tumor-suppressors in non-CESC cancers and their roles in CESC were unclear. This study provided experimental directions for these novel genes and miRNA.Some limitations of the current study should be noticed. (1) For PH assumption test, it is difficult to estimate the type II error . Thus, it is hard to choose a threshold of PH assumption test We have identified a novel transcriptomic signature for CESC prognosis, including 1 miRNA, 1 lncRNA and 6 mRNAs. The transcriptomic signature was more comprehensive and predictive than the miRNA-only, the gene-only, and the previously identified signatures for CESC prognosis.JX conceived and designed the analysis. JX, SG, YS, LG, and ZB performed the analysis. JX wrote the paper.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.


Loss of motor function is a common deficit following stroke insult and often manifests as persistent upper extremity (UE) disability which can affect a survivor’s ability to participate in activities of daily living. Recent research suggests the use of brain–computer interface (BCI) devices might improve UE function in stroke survivors at various times since stroke. This randomized crossover-controlled trial examines whether intervention with this BCI device design attenuates the effects of hemiparesis, encourages reorganization of motor related brain signals (EEG measured sensorimotor rhythm desynchronization), and improves movement, as measured by the Action Research Arm Test (ARAT). A sample of 21 stroke survivors, presenting with varied times since stroke and levels of UE impairment, received a maximum of 18–30 h of intervention with a novel electroencephalogram-based BCI-driven functional electrical stimulator (EEG-BCI-FES) device. Driven by spectral power recordings from contralateral EEG electrodes during cued attempted grasping of the hand, the user’s input to the EEG-BCI-FES device modulates horizontal movement of a virtual cursor and also facilitates concurrent stimulation of the impaired UE. Outcome measures of function and capacity were assessed at baseline, mid-therapy, and at completion of therapy while EEG was recorded only during intervention sessions. A significant increase in r-squared values [reflecting Mu rhythm (8–12 Hz) desynchronization as the result of attempted movements of the impaired hand] presented post-therapy compared to baseline. These findings suggest that intervention corresponds with greater desynchronization of Mu rhythm in the ipsilesional hemisphere during attempted movements of the impaired hand and this change is related to changes in behavior as a result of the intervention. BCI intervention may be an effective way of addressing the recovery of a stroke impaired UE and studying neuromechanical coupling with motor outputs.Clinical Trial Registration:ClinicalTrials.gov, identifier NCT02098265. Stroke is a leading cause of acquired adult long-term disability in the United States  and occuSeveral rehabilitation techniques are traditionally used for stroke recovery including conventional physical-occupational-speech therapies, provided in acute care settings as well as newer motor therapies such as constraint-induced movement therapy (CIMT), robot-aided therapy, transcranial direct current stimulation (tDCS), transcranial magnetic stimulation (TMS), and virtual reality (VR) . ImportaResearch suggests that motor recovery post-stroke, similar to motor learning, requires specific internal and external environmental conditions . For exaHuman brain rhythms associated with motor output, sensorimotor rhythms (SMRs), are recorded superficial to the motor and somatosensory cortical strip of the brain (electrode sites C3 and C4) and originate according to homuncular organization . At the Mu and Beta sensorimotor rhythms (SMRs) in human subjects are recorded exclusively over sensorimotor areas at frequencies of about 10–20 Hz . Two basNoninvasive brain–computer interfaces (BCIs), which utilize ancillary adjuvant peripheral devices and electrical muscle stimulation, as well as invasive BCI approaches with electrodes implanted in the skull, have been introduced  as conteIn this study, EEG driven BCI was linked to tongue stimulation (TS) via a Tongue Display Unit (TDU)  designe reinforcGrowing evidence from our lab  and otheIt was hypothesized that (1) the EEG-BCI-FES intervention sessions would result in increased hemispheric desynchronization levels of Mu (8–12 Hz) rhythm and, or Beta (18–26 Hz) band signals over the ipsilesional motor cortices, as reflected by increased r-squared values , and (2) changes in functional connectivity (coherence) are greatest in the affected contralateral  motor cortex and, over time, are associated with beneficial behavior and quality of life improvements as measured by objective and subjective measures of upper extremity motor function and activities of daily living. This interim analysis, of the larger ongoing prospective randomized crossover-controlled clinical trial, seeks to determine whether greater desynchronization of motor related SMRs in the ipsilesional hemisphere during attempted movements of the impaired hand are related to changes in behavior as a result of intervention.Participants were recruited from the greater Madison, WI, United States area as part of an on-going prospective randomized, cross-over controlled design stroke rehabilitation study investigating interventional BCI targeting UE motor function. This study is approved by the University of Wisconsin Health Sciences Institutional Review Board (Study ID 2015-0469); all subjects provided written informed consent upon enrollment. A CONSORT flow diagram is made available in ClinicalTrials.gov (study ID1 NCT02098265), employs an open call for participants with a wide range of (1) UE hemiparesis resulting from stroke, (2) time-since-stroke, (3) stroke type, (4) lesion location, (5) number of previous strokes, (6) and stroke severity. Subsequent to informed, written consent, stroke survivors were randomized by permuted-block design accounting specifically for gender, stroke chronicity, as well as severity of motor impairment as measured by the Action Research Arm Test (ARAT) . Chronicity is measured as time since stroke, in days, to baseline measurement day. Participant characteristics are displayed in This on-going study, registered with t (ARAT)  , psychiatric (affect), or developmental disabilities were included. Exclusion criteria were: allergy to electrode gel, surgical tape, metals, concurrent treatment for infectious disease, apparent lesions or inflammation of the oral cavity, pregnancy or intention to become pregnant during the course of the study, or any contraindication for magnetic resonance imaging (MRI). Subjects from the greater study cohort were excluded from the presented analyses if they (1) failed to complete at least 9 of 15, two-hour BCI intervention sessions occurring at least twice each week, (2) failed to complete all four MRI and behavioral testing sessions occurring in the intervention phase .Participants, when assigned to the intervention phase, have at least 9 and up to 15 EEG-BCI-FES intervention sessions (two-to-three sessions/week) wherein they receive EEG-BCI-FES intervention lasting up to 2 h for a potential total dosing of 30 h of EEG-BCI-FES intervention. Along with the EEG-BCI-FES intervention sessions, subjects also receive fMRI and behavioral testing at four time points: prior to the first EEG-BCI-FES intervention session (baseline), after the first few weeks of intervention (midpoint), following the final intervention session (endpoint), and again 1 month after the endpoint assessment (follow-up). Subjects assigned to the delayed intervention group (DTG) are encouraged to continue with their normal and customary care while in the delay period. While in the delay period, participant EEG data are not recorded and participants are instructed not to use a BCI device. During this time, there are three assessment visits consisting of MRI and behavioral testing which are matched in sequence and duration to those conducted in the BCI intervention period as demonstrated in The BCI system and intervention sequence were consistent with those previously described ,b,c usinSubjects went through intervention sessions on separate days. The number of EEG-BCI-FES intervention sessions varied across subjects with a mean of 13.8 ± 1.3. Each EEG-BCI-FES intervention session consisted of multiple runs of the ‘Cursor Task’ (mean of 31.3 ± 10.5 runs per session), about 1/3rd of which included only visual feedback, and roughly two thirds of which were comprised of BCI facilitated functional electric stimulation of the impaired hand and lingual electrotactile stimulation through a tongue display unit (TDU)  Figure . The EEGThe first BCI session was focused on assisting the participant to comprehend and engage the BCI device and protocol. Stroke survivors often present with a myriad of cognitive, affective, and physical impairments  and out EEG data were recorded using a 16-ch bioamplifier  from 16 active electrodes using a g.GAMMA cap   accordin2. The main processing steps for the EEG data collected during screening phases were as follows:The raw EEG data files were loaded into Fieldtrip , and fully processed using tools incorporated in this toolbox  and MATL2 for low and high variance signals, respectively) and repair of bad (noisy) channels (spline interpolation), followed by the removal of three trials showing the highest variance  Digital filtering with a high-pass filter cutoff of 4 Hz, and a low-pass filter cutoff of 30 Hz. (2) Extraction and grouping of trials according to condition , movement type , and the screening phase . This resulted in 10 trials for each of condition/movement/screening phase combinations. (3) Identification  Calpriori hypotheses of expected changes in the r-squared values and coherence as the result of intervention time at C3 and C4 sites were tested using paired t-tests in MATLAB. Associations between the r-squared changes and the total number of intervention runs as well as behavioral changes  were assessed using Pearson’s and Spearman’s correlation, respectively. Finally, the associations between the signed r-squared values with behavioral scores from several tests at baseline were assessed using Spearman’s or Pearson’s correlation coefficients, as appropriate. Thresholds for significance and trend toward significance were set a priori at p ≤ 0.05 and 0.05 < p < 0.1, respectively, for all statistical analyses.The independent variables were the signed r-squared values and the coherence estimates. At individual subject level, the data consisted of average estimates per each session for condition/movement/screening phase combination sets, and at group level the estimates consisted of grand averages over sessions of each individual subject data in the group (pre- and post-therapy scores averaged separately across sessions). Non-parametric statistical tests were run by calculating Monte-Carlo estimates of the significance probabilities and critical values from the permutation distribution , followeThe primary outcome measure was the ARAT. The ARAT is a 57-point test designed to assess specific changes in upper limb function with sub-components for grasp, grip, pinch, and gross motor movement . The sect-test to evaluate the statistical significance of ARAT and secondary outcome measure changes  between baseline, completion, and follow-up scores , of which nine (64%) realized improved scores after intervention, both at immediate completion and 1 month after completion. Participant characteristics are summarized in p = 0.046), and change at follow-up (p = 0.020) (p = 0.046). This particular finding did not persist at the 1-month follow-up time point. Statistical significance was observed in the baseline to follow-up score analyses (p = 0.05)  : T7, cha = 0.05) . All staResults reported below in Section “R-Squared” echoed in the graph in t-test: t(20) = 1.85; p = 0.039; meanPRE = -0.142; meanPOST = -0.161], while the subject attempted movements of the impaired hand (t-test: t(20) = 1.24; p = 0.114; meanPRE = -0.131; meanPOST = -0.145]. The signed r-squared value  for the Mu (8–12 Hz) rhythm significantly decreased in the post-therapy stage compared to the pre-therapy stage  while the subjects attempted movements of the impaired hand, although this change did not achieve statistical significance  values, calculated from coherence estimates at C3 and C4 sites from Beta band (18–26 Hz) signals, decreased in post-therapy stage compared to the pre-therapy stage   in the ipsilesional hemisphere motor site during impaired hand attempted movements, following the intervention, showed a significant correlation with the total number of cursor trials  runs [= 0.043] . Item nup = 0.098]  in the ipsilesional hemisphere motor site during impaired hand attempted movements, as the result of EEG-BCI-FES intervention, showed a positive, non-statistically significant correlation with the change in ARAT scores (obtained post-therapy in comparison to baseline)  .Finally, to test some of the fundamental assumptions of the study design and BCI device (that diminished SMR desynchronization is related to the post-stroke impairment of simple motor outputs), signed Mu and Beta r-squared values for the impaired hand attempted at baseline  were compared to measures of behavior , and measures of stroke-related impairments to functional capacities  at baseline . A few mNo adverse events were reported during or after participation in the research experiment.The findings that motor cortex EEG measures during attempted movements of the impaired hand  are positively correlated with behavioral changes and seem to offer a measurable link between electrophysiology and behavior is in line with the hypotheses set forth in this analysis. More importantly, the significant group level changes in r-squared values post-therapy compared to pre-therapy suggest an effect of the applied EEG-BCI-FES intervention protocol which may be beneficial for motor recovery, though data are currently inconclusive. As stated in Section “Amount of Intervention: Number of Runs,” the amount of BCI practice was positively correlated with Beta band ERD of the ipsilesional motor cortex. Thus, it may be possible to conceive that, following adequate amounts of training; electrophysiological measures of connectivity such as coherence may allow additional insights into the potentials and mechanisms of functional change to the neuromuscular and neuromechanical coupling of effortful motor movement.A strength of this design and analyses for evaluation of objective physiological or functional changes as the result of the EEG-BCI-FES intervention is that the EEG-based measures extracted and compared were obtained immediately before, and immediately following each BCI intervention session , at the pre an post screening periods . By compThese results suggest that EEG-BCI-FES has the potential to induce neuroplastic change and aid recovery of UE paresis. However, this analysis was constrained by sample size and heterogeneity in lesion location, level of impairment, and time since stroke. Greater power is needed to adequately generalize these results. Utilizing a larger and more homogeneous subject cohort could allow for more generalizable conclusions in future research. Further, 16 electrodes were used in EEG signal data acquisition and EEG were recorded only during the intervention phase and at no other time during the study. While there is no EEG data recorded in the control period to compare with the recordings during intervention, there are brain (EEG) – behavior correlations specifically in EEG measures associated with motor function originating specifically from electrodes (C3/C4)  overlyinIt is generally understood that using 16 electrodes is insufficient for source localization, especially given the limited spatial coverage and non-equidistant spacing of the electrodes in this cap array  and thusSuch heterogeneity and restricted sample size similarly dissuaded the authors from attempting further conservative controls, such as multiple comparisons corrections. The authors conceived that further conservative data manipulations may wash out any potential (‘trending to’) significant relationships the authors or other groups may want to follow-up with future research. This manuscript, part of a larger on-going clinical trial, is an interim analysis which sought to elucidate any significant trends in the data as the study progressed so as to inform our future questioning of the data and to be better prepared to identify and test potentially significant interactions and factors in the larger post-stroke population.This is an on-going study in its seventh year of data acquisition and participant enrollment. Various project personnel have undergone and supervised the staffing, training, and data acquisition of this trial during its course. The authors work hard to best minimize differences in acquisition of study measures through extensive and repeated training of personnel.Despite the existing challenges to providing evidence-based treatment strategies in the stroke rehabilitation field, combined therapies may be used to achieve the maximal motor function recovery for participants . DevelopNon-invasive EEG-based measures of motor cortex function, such as r-squared (reflecting desynchronization levels of the relevant SMR rhythms), could provide an efficient means of tracking and even predicting functional changes in stroke patients during the course of the EEG-BCI-FES intervention. As ERD changes were reported at the group level, and given the heterogeneity in the sample, it may be argued that the reported changes not only suggest a change in function for the majority of participants  but also, given more selective sampling and independent variable control, an even more clinically relevant relationship between ERD and recovery may exist. Tracking SMR modulations may be a potential predictor of recovery or indicator of recovery potential in a patient.The observed effects to motor measures might also be a consequence of challenging and rewarding movements associated with (ADLs), which the participants previously may have thought to be impossible or too difficult to produce successfully. BCI intervention may help challenge a survivor’s individual conception of their limitations by pushing a participant to use the affected hand and rewarding them  for doing so. This is to suggest that the minimal gains observed by most participants, in comparison to the significant gains obtained by some, and their absence in others, may be related to the encouragement of attempting previously ineffective motor behaviors. It is possible the statistically significant gains observed, supported by the higher incidence of significance in subjective measures than the number of lab-based objective measures, could be the result of the specific reward structure of the design in addition to, or more so than any reliable neuromechanical or electrophysiological contributions.If normal muscle synergies  are disrupted by an insult such as stroke, robbing the motor circuity of its primary output components , residual functional capacities are limited by the ability of the system to retrain or re-map (link) the CNS commands to PNS effectors . SuccessFrom previously published findings , we can Losing strategies, more often than not, do not win . Maladaptive associations may simply need more time to be pruned away and relevant adaptive associations strengthened by increased and more highly structured reinforcement . If one AR and LW contributed equally to the writing of this manuscript. AR was involved in subject recruitment, staff training, data collection, data processing, data analysis, interpreting data, and writing of the manuscript. LW was involved in staff training, data collection, data processing, data analysis, interpreting data, and writing of the manuscript. KG was involved in analysis, interpreting and writing of the manuscript. KD, JT, TJ, were involved in data collection, analysis, and editing the manuscript. BY was involved in subject recruitment, staff training, data collection, data processing, data analysis, interpreting data, intellectual content, and manuscript editing. ZN was involved in subject recruitment and data collection. MW, MMc, MMa, SR, HA, RM, NT, JA, and LW were involved in data collection. VN contributed to subject recruitment, staff training, data collection, data processing, data analysis, interpreting data, manuscript editing, and intellectual content. PVK contributed to manuscript editing and intellectual content. JS was involved in recruitment of study participants, study design, and intellectual content. TK was involved in the recruitment of study participants, manuscript editing, and intellectual content. JCW, DE, and VP are co-PIs and were involved in study conception, design, staff training, manuscript editing, intellectual content, and supervised all aspects of the study.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.


Worldwide, roughly 80% of adolescents fail to meet World Health Organization (WHO) recommendations regarding physical activity, though there is substantial variation in adolescent physical activity prevalence across countries. This study explored whether country-level environmental differences explained cross-national variation in adolescent moderate- to vigorous-intensity physical activity (MVPA) and vigorous-intensity activity (VPA).Using the data of 138,014 11- to 15-year-olds from 29 European countries in the 2013/2014 Health Behaviour in School-aged Children (HBSC) study, multilevel regression models examined the influence of four types of country-level environmental factors  on self-reported individual-level physical activity (MVPA and VPA).The environmental variables explained 38% of country-level variance in MVPA and 81% of country-level variance in VPA. Lower annual average national temperature, higher community safety, lower average national household income and a weaker physical education policy were significantly associated with more MVPA. Greater urbanisation, lower annual average national temperature, higher adult physical activity and higher average national household income were significantly associated with more VPA.The findings showed that national differences in the physical, socio-cultural and economic environment were related to adolescent physical activity. They point to potential avenues for future research looking at interactions between individual and environmental factors.The online version of this article (10.1186/s12889-019-6908-9) contains supplementary material, which is available to authorized users. Physical inactivity in adolescence is a critical public health issue . The WorHowever, levels of inactivity in adolescents and adults are high and estimated to cost $67.5 billion worldwide through health-care expenditure and productivity losses . In 2010Importantly, substantial variation in adolescent physical activity prevalence exists across and within countries , 14, 15,International variation can be attributed to differences in the characteristics of individuals within a country, such as adolescent motivations for participation in physical activity . Motivatphysical environment to be important for physical activity. Urbanisation contributes to land-use mix ; systemkability , 27. Temkability . Seasonakability .socio-cultural environment  is better understood at the micro (e.g. home and school) level than at the macro  level ) Despite considerable cross-national variability in adolescent physical activity, explanations of this variability are scarce. Previous research examining the role of environmental factors has given limited attention to the relative importance of different types of environmental factors, so this study examined eight different factors in a single design. The findings that physical, socio-cultural, economic and political environment factors are all related to physical activity provide support for ecological theory and emphasise the importance of taking different environmental factors into account simultaneously. Future research should acknowledge the conceptual difference between MVPA and VPA, given they are associated with different environmental factors. Intervention planning must therefore consider how environmental strategies affect both moderate and vigorous physical activity. Notwithstanding the effects we found on the country-level, effects on the individual level – where most of the variation in adolescent physical activity takes place – are much stronger. However, from a scientific and public health perspective it is important to know how the country-level variance can be explained. Further studies could examine mechanisms through which country-level factors affect adolescent physical activity or look more closely at interactions between individual and environmental factors, which might shed light on cross- national gender, age and socio-economic differences . This maAdditional file 1: Information on ethical approvals in the HBSC study 2009/2010, provided by the principal investigator of each country. (DOCX 16 kb)