Background: With the growing momentum for the adoption of machine learning (ML) in medical field, it is likely that reliance on ML for imaging will become routine over the next few years. We have developed a software named BAAD, which uses ML algorithms for the diagnosis of Alzheimer's disease (AD) and prediction of mild cognitive impairment (MCI) progression.Methods: We constructed an algorithm by combining a support vector machine (SVM) to classify and a voxel-based morphometry (VBM) to reduce concerned variables. We grouped progressive MCI and AD as an AD spectrum and trained SVM according to this classification. We randomly selected half from the total 1,314 subjects of AD neuroimaging Initiative (ADNI) from North America for SVM training, and the remaining half were used for validation to fine-tune the model hyperparameters. We created two types of SVMs, one based solely on the brain structure (SVMst), and the other based on both the brain structure and Mini-Mental State Examination score (SVMcog). We compared the model performance with two expert neuroradiologists, and further evaluated it in test datasets involving 519, 592, 69, and 128 subjects from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Aging (AIBL), Japanese ADNI, the Minimal Interval Resonance Imaging in AD (MIDIAD) and the Open Access Series of Imaging Studies (OASIS), respectively.Results: BAAD's SVMs outperformed radiologists for AD diagnosis in a structural magnetic resonance imaging review. The accuracy of the two radiologists was 57.5 and 70.0%, respectively, whereas, that of the SVMst was 90.5%. The diagnostic accuracy of the SVMst and SVMcog in the test datasets ranged from 88.0 to 97.1% and 92.5 to 100%, respectively. The prediction accuracy for MCI progression was 83.0% in SVMst and 85.0% in SVMcog. In the AD spectrum classified by SVMst, 87.1% of the subjects were Aβ positive according to an AV-45 positron emission tomography. Similarly, among MCI patients classified for the AD spectrum, 89.5% of the subjects progressed to AD.Conclusion: Our ML has shown high performance in AD diagnosis and prediction of MCI progression. It outperformed expert radiologists, and is expected to provide support in clinical practice. Approximately 50 million people have dementia worldwide, with almost 10 million new cases annually. Alzheimer's disease (AD) is the most common form of dementia and contributes to well over 50% of cases. In machine learning (ML), data are transferred by functions into a high-dimensional mathematical space to properly form clusters. The number of dimensions corresponds to the number of concerned variables, and ML enables the handling of a large number of variables to analyze the data. In high-dimensional space, it is easier to find a separable boundary. However, as the dimension increases, the data become sparse, and more data are needed to obtain an optimal solution. This is known as the “curse of dimensionality.” In situations where the amount of available data is limited, it is necessary to devise an appropriate way to incorporate the configuration of concerned variables to reduce the number of variables used. If the data scientist is familiar with the meaning of the dataset and its numerical features, important variables can be selected intentionally. An alternative method is using prior operations, such as voxel-based morphometry (VBM), which normalizes the brain shape and scales the regional brain volumes by adjusting for brain size, age, gender, etc. Selecting which ML algorithm to use is crucial, as each has its own strengths and weaknesses. For example, when analyzing a large amount of data, deep learning (DL) often outperforms other alternative ML algorithms through representation learning and self-optimization. However, the learning process of DL cannot be fully interpreted; it is difficult to validate the results, and the preparation of a large amount of data is a practical challenge. When the data size is small , a support vector machine (SVM) can perform well when combined with prior operations such as VBM. As is often the case with medical image data, if the available data are small, it is effective to standardize the data by VBM prior to machine learning. Here, we introduce a software named brain anatomical analysis using diffeomorphic deformation (BAAD) that enables image analysis with ML, combining VBM and SVM for the diagnosis of Alzheimer's disease (AD) and prediction of mild cognitive impairment (MCI) progression to AD.Hippocampal atrophy is one of the diagnostic biomarkers for AD; however, how do clinicians objectively evaluate it see . There ahttp://www.shiga-med.ac.jp/~hqbioph/BAAD(English)/BAAD.html) also uses VBM but includes algorithmic analysis not found in VSRAD. In the case shown in z-scores between VSRAD and BAAD was partly owing to the differences in VBM procedures and ROI location, but mainly because of the statistical design. The mean age of the reference group provided by VSRAD was approximately 70 years, which is relatively higher than the subject's age of 58 years, resulting in underestimation of brain atrophy of the ROIs. BAAD uses age as a covariate, resulting in age corrected z-scores of the hippocampi that are more accurate for the subject's age. As shown in this example, when assessing hippocampal atrophy, we may unknowingly compare the size with that of a subject of approximately 70 years old, which is common in AD patients. Moreover, the ML in BAAD showed an AD likelihood score of 0.944 (max = 1), indicating high probability of AD. This ML predicts AD based on information in entire brain regions as well as the hippocampus.The BAAD software developed in this study  software, as first proposed by Ashburner et al. . The floCoordinate transformation from native space to MNI space was performed using Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) algorithm . This ishttp://marsbar.sourceforge.net) to perform ROI-wise analysis using preset ROIs for automated anatomical labeling (AAL), Brodmann's atlas, and the LONI Probabilistic Brain atlas (LPBA40). Each atlas has ROIs of 108, 118, and 56, respectively. In this process, local volume was adjusted by total intracranial volume (TIV) and age. We did not include sex as a covariate, as it was empirically known that sex differences were offset by TIV. For z-score estimation, BAAD used subjects over age 50 in the IXI database as a reference, with age and TIV as confounding factors. Z-scores were derived from /(control standard deviation).To reduce the number of feature vectors for the SVM classifier, BAAD uses multiple sets of adaptive ROIs instead of the voxel-wise method . There aPr = (Y = k|X = x), where the probability Y is the class k given that the input variable X is x. The probability is transformed by a sigmoid function so that the value is within the range of . The larger the value, the higher the likelihood of AD. We used radial basis function (RBF) for the SVM kernel, and the values of parameters were optimized using the Alzheimer's disease neuroimaging initiative (ADNI) database. The basic principal of SVM is explained in the Supplementary Section. For training and validation, we introduced a spatial-anatomical method by providing the z-score of the ROIs as a feature vector. Leave-one-out cross validation was used to obtain unbiased estimates and to avoid over-fitting in this procedure. A mathematical procedure was performed using the “fitcsvm” function implemented in MATLAB R2016b . The sequential minimal optimization (SMO) algorithm and “bayesopt” were used for the optimization of the parameters (We expressed the likelihood of the AD brain as the Alzheimer's disease likelihood score (ADLS), which represents the distance to the separating hyperplane. ADLS is obtained from the posterior probability function, rameters . Finallyhttp://www.adni-info.org/Scientists/AboutADNI.aspx).We used ADNI data from three countries; North America (NA-ADNI), Australia ), and Japan (JADNI). Data from North America and Australia were extracted from the LONI Image and DATA Archive (ida.loni.usc.edu). Data from Japan were extracted from the National Bioscience Database Center (hum0043.v1). More information about the database can be found at adni-info.org. The NA-ADNI was launched in 2003, and started in 2004 as a longitudinal, multicenter cohort study of healthy elderly and individuals with MCI and early AD. AIBL and JADNI were launched in 2006 and 2007, respectively. More information on the diagnostic criteria can be found on the ADNI web site . OASIS provides freely available data and the details are available elsewhere , 544 MCI and 359 AD participants. We classified MCI patients into progressive MCI (pMCI) and stable MCI (sMCI), depending on the progression to AD or not. Of the sMCI cases, 132 subjects were excluded because of the short observation period (<4 years), which will be discussed in more detail later in the paper. The training set consisted of 723 randomly selected subjects, and the remaining 723 subjects created the validation set . To verilsewhere .Structural MR images of the brain were acquired with 1.5T  and 3.0T  scanners from several vendors including Philips Medical Systems, Siemens and GE healthcare. All images were scanned under the ADNI protocol conditions with 3D-sagittal plane slices and magnetization prepared rapid acquisition gradient echo sequence (MPRAGE) sequence . In the MCI is a transitional stage between normal aging and dementia. It is estimated that between 5 and 20% of people over 65 years of age have MCI, and the annual rate of progression to AD is 10–15% among persons with amnestic MCI . The 3-yAD can be conceptualized as a biological and clinical continuum from the preclinical phase  to the clinical phase. Therefore, the preclinical and clinical phases are a seamless sequence. Diagnosis of MCI can be accomplished according to prescribed criteria at the research level, but it may be difficult to implement the same complex protocols in clinical practice. Therefore, it is better not to dwell on the distinction between MCI from normal (NL) or AD. For this reason, we labeled AD and pMCI as being in the AD spectrum, and NL and sMCI as being in the non-AD spectrum for SVM learning. We randomly extracted 321 AD spectrum (179 AD and 142 pMCI) and 335 non-AD spectrum images from the NA-ADNI database (271 NL and 64 sMCI) , and usewww.mricro.com). Third, after completion of the initial diagnosis, the radiologists were allowed to modify their diagnoses with reference to the results of VSRAD.Two neuroradiologists (RI and HK) independently and blindly reviewed the structural MR images. Both neuroradiologists are board-certified experts in Japan with more than 20 years of clinical experience. MR images of 100 AD and 100 NL subjects were randomly selected from the NA-ADNI database, and any information other than age and gender of the subjects was not disclosed to the radiologists. First, 10 AD and 10 NL subjects were extracted from the sets and used for training. Second, a few days later, the radiologists were asked to diagnose AD or NL from the 200 structural images using MRIcron software . To define four cortical gray matter regions , brain MRI for each subject was segmented and parcellated with FreeSurfer (version 5.3.0). The standardized uptake value ratio (SUVR) was calculated by creating a conventional (non-weighted) average across the four main cortical regions and was normalized by the entire cerebellum reference region. Data were downloaded from the LONI web site (https://ida.loni.usc.edu/login.jsp). The SUVR cutoff value was defined above 1.11 according to the recommendation of UC Berkeley  of p < 0.05.Kaplan-Meier and other statistical analyses were performed with JMP® software . The receiver operating characteristic (ROC) curve was used to evaluate the ability of the model for classifying disease. To obtain maximum potential effectiveness, optimal cut-off point was determined by Youden Index. The equations of accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), F1, and Matthews correlation coefficient (MCC) are available elsewhere , 20. MCCThe results of the ML performance for AD diagnosis in the NA-ADNI . The kappa coefficient, an index of the degree of concordance between the two radiologists was as low as 0.35  without VSRAD, but it improved to 0.56 after referring to VSRAD .The diagnostic accuracy of the two radiologists who reviewed 200 subjects selected from the NA-ADNI database was 57.5 and 70.0%, which was lower than the SVMst at 90.5% . After dTo assess the ability of the ML to predict MCI progression, we analyzed data from 142 pMCI and 64 sMCI subjects from the NA-ADNI validation set, and the results are summarized in We assessed the predictive accuracy of the MCI progression by SVMst in the NA-ADNI database for each timing of MRI examinations before the onset of AD and compared it to the predictive accuracy of AV-45 PET . As expen = 103) of the Aβ-positive MCIs (n = 113) was 91.2%, or 92.8% of all pMCI (n = 111). In addition, 8 of 11 (72.7%) SVMst-positive MCI patients progressed to AD, even though they were AV-45 negative. For SVMst-negative, 47.1% of the subjects were Aβ positive, and pMCI (n = 15) accounted for 23.8% of the total MCI (n = 63). When restricted to Aβ-negative MCIs (n = 57), the percentage of pMCI (n = 6) was 10.5%. These results suggest that there is a strong association between Aβ accumulation and brain atrophy in AD-like patterns. Note that 11.7% patients clinically diagnosed with AD were Aβ negative. These patients may later become Aβ positive, or if not, this indicates the difficulty of AD clinical diagnosis.The relationship between ADLS and Aβ deposition was investigated in 771 subjects from an AV-45 PET study because Aβ is one of the important molecular targets for disease modifying therapies (DMT). When classified as positive (ADLS > 0.5) by SVMst, 87.1% of the subjects were Aβ positive (SUVR > 1.11) . In SVMsThis study showed that the proposed BAAD software provides a useful opportunity to address the diagnosis of AD and prediction of the disease progression in MCI patients. A strict classification between MCI and AD is not always necessary in daily medical care; therefore, the SVM was trained by grouping pMCI and AD as the AD spectrum. Subjects assigned to the AD spectrum by the ML are at high risk of having AD pathology, even if their cognitive decline is not significant. Furthermore, by using the MMSE score, SVMcog classified AD and NL at accuracies ranging from 93.8 to 100% in the test databases. In the SVMst-positive cases, 111 of 124 MCIs (89.5%) progressed to AD, 113 (91.1%) were Aβ positive on AV-45 PET, and 103 (91.2%) progressed to AD. Interestingly, for MCI patients with negative AV-45, 72.7% progressed to AD if the SVMst classified them as being in the AD spectrum. It is debatable whether any patient who has AD pathology is suitable for DMT. However, it is helpful to predict patients at high risk of AD progression to facilitate more disease-specific examinations such as amyloid or tau PET, depending on the target of DMT.It requires some consideration to apply the proposed model to clinical use because the subjects in the ADNI database have already been selected and simplified by excluding other types of dementia. For example, we need to be aware that the ML is not designed to distinguish AD from other types of dementia. In clinical practice, AD is not diagnosed by MRI alone, rather it is used to exclude brain infarction, tumors, infection, hydrocephalus, and other diseases. If there is any doubt, regional cerebral blood flow (CBF) imaging by single photon emission computed tomography (SPECT) has diagnostic value, particularly in differentiating AD from frontotemporal dementia , 22. DopThe BAAD ML outperformed radiologists in the diagnostic accuracy of AD for reviewing structural MRI. VSRAD reduced inconsistency between the two radiologists and improved the diagnostic accuracy. However, the mean diagnostic accuracy of the radiologists was 71.5% even with the support of VSRAD, whereas that of SVMst was 90.5%. The reason for this is that VSRAD relies only on the volume of the medial temporal structures, whereas the SVMst integrates the information from many ROIs covering the entire brain. Age correction for VBM statistics is also a strength of BAAD, as shown for an illustrative case in In our model, all MR images were preprocessed by VBM to standardize and simplify the features. Specifically, this included ensuring a unified brain shape, converting the volume into signal intensity, and adjusting the brain volume according to TIV and age. Furthermore, the number of features was reduced by using ROIs instead of individual voxels. Unlike other organs, the brain forms a neuronal network and functional localization, and tau pathology of AD are believed to propagate along the neural network . We adopA recent study of longitudinal tau PET showed that Aβ accumulation promotes tau pathology, and the tau accumulation rate was increased in the entorhinal, fusiform, inferior and middle temporal, temporal pole, retrosplenial, and the posterior cingulate cortices in the early stage of the disease. In cognitively impaired individuals, tau accumulation was found in all regions, but higher rates were in the inferior temporal, superior orbitofrontal, basal frontal (olfactory and gyrus rectus), and middle occipital cortices . The stuThere are several limitations to this study. First, as is often pointed out in studies using the ADNI database, patients with non-AD dementia are carefully excluded in advance. Therefore, the clinical validity of the AD diagnosis by the model in this study is limited when patients are screened out for other forms of dementia. Second, the definition of sMCI in our study depends on the follow-up period of the study. For example, some patients with MCI may eventually progress to AD if they have a longer follow-up period. Therefore, it is not recommended to use this ML for long-term preventive treatment. It is not possible to accommodate the non-amnestic type because the MCI patients are weighted to the amnestic type in the ADNI study. Third, once the brain structure of the subject is shared by both the training and test datasets, it becomes difficult to articulate model overfitting. We confirmed the performance of the ML on several untrained databases, but this was limited to the case of AD diagnostics. The accuracy of the pMCI prediction in JADNI was the result of replacing sMCI with NL; therefore, a future goal is to accumulate observed data on sMCI over a long period.For a patient with memory disturbance or suspected dementia, it is expected that the proposed ML may support clinicians to diagnose or predict the progression to AD, while the possibility of dementia other than AD should always be kept in mind. For patients with clinically suspected AD, the SVMcog supports AD diagnosis with 95% accuracy, based on MRI and MMSE. Approximately 90% of MCI patients were Aβ-positive when classified as AD spectrum by SVMst. Therefore, these patients should be closely monitored for 3 years or more. Molecular PET examinations will be considered for DMT applications in the future. Currently, there are no codified indicators to accurately predict pathological progression in the prodromal stages owing to the contradictory results regarding the concentration of CSF biomarker proteins . Based oThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.AS was responsible for the project administration, supervision, writing–review and editing. AHS contributed to the data collection, management, and analyses. HK and RI participated in the AD diagnosis of MR images. MI and KT contributed to the research design. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An ad hoc questionnaire was prepared to collect sociodemographic and work data, and the Maslach Burnout Inventory, the Perceived Stress Scale, the Font–Roja Questionnaire, the Brief Cope Orientation to Problem Experience and the General Health Questionnaire were used. The prevalence of high burnout was 8.19%. The levels of perceived stress and job satisfaction were moderate. The most frequent clinical manifestations were social dysfunction and somatic symptoms, and problem-focused coping was the strategy most used by nurses. Lack of physical exercise, gender, years worked at an emergency department, anxiety, social dysfunction, and avoidance coping were significant predictors of the dimensions of burnout.Burnout is a major problem among nurses working in emergency departments and is closely related to a high turnover of personnel, nursing errors, and patient dissatisfaction. The aims of this study were to estimate burnout, perceived stress, job satisfaction, coping and general health levels experienced by nurses working in emergency departments in Spain and to analyze the relationships between sociodemographic, occupational, and psychological variables and the occurrence of burnout syndrome among these professionals. A cross-sectional study was conducted in four emergency departments in Andalusia (Spain) from March to December 2016. The study sample was composed of  The three key dimensions of this response are: (i) emotional exhaustion (EE), or a loss of enthusiasm in one’s work; (ii) depersonalization (DP), or an impersonal response to patients; and (iii) personal accomplishment (PA), or a feeling of carrying out one’s job successfully ,2. Burnoenomenon  affectinenomenon . Healthcenomenon , and nurenomenon . Studiesenomenon ,8.However, the various clinical contexts differently affect the nurses’ susceptibility to burnout ,10,11. IEmergency departments (EDs) are experiencing increasing service demands ,16, whicEqually as troubling is job dissatisfaction. High levels of stress and burnout are linked to lower satisfaction in nursing professionals . Job satAlthough occupational stress and burnout are common among ED nursing personnel, not all ED nursing staff show altered levels of the aforementioned dimensions. Some professionals will thrive in the same seemingly stressful environment. One important factor that may influence the likelihood of job stress and burnout is an individual’s coping style, defined as the cognitive and behavioral efforts applied by an individual to manage stress . ResearcDespite occupational stress, job satisfaction, and coping styles are associated with general health of nurses, burnout is most significantly associated with general health , resultiTo the best of our knowledge, this study is the first research in ED nurses that measures and compares these different types of psychological states at the same time. It is important to identify the most significant relationships between occupational stress, burnout, coping, job satisfaction, and general health so that evidence-based policy and practice aimed at promoting healthy working environments for nurses can be promptly developed. The main objectives of this study were therefore as follows: to estimate the burnout, perceived stress, job satisfaction, coping and general health levels experienced by nurses working in Spanish EDs and to analyze the relationship between the sociodemographic, occupational, and psychological variables and the occurrence of burnout syndrome among these professionals.A quantitative, observational, cross-sectional, multicenter study.The study was carried out at the EDs of four hospitals in Andalusia (southern Spain). ED 1 has 165,520 visits per year and a reference population of 481,296 inhabitants; ED 2 has 135,000 visits per year and a reference population of 461,078 inhabitants; ED 3 has 48,000 visits per year and a reference population of 62,234 inhabitants; ED 4 has 18,000 visits per year and a reference population of 24,287 inhabitants.In order to assess the suitability of the study population, the required sample size was calculated using Epidat version 3.1 . We considered a 95% confidence level, an absolute precision of 3%, and a prevalence of burnout among ED nursing staff of 26% . With th212 cover letters with the questionnaires were sent in sealed envelopes to all the nurses in the four EDs selected. This number comprised the total number of nurses employed by the four EDs at the start of the study which complied with the inclusion criteria, which included all the active nurses during data collection who had worked at the ED for at least one year. The exclusion criteria were nurses on sick leave or unpaid leave during data collection. In the end, 171 questionnaires were completed (80.66% response rate).The data were collected from March to December 2016. The study data were compiled for the following sociodemographic, occupational, and psychological variables: sociodemographic variables, including sex , age (years), marital status , daily physical exercise , and daily tobacco use . The occupational variables included type of employment contract , time of service at the ED (years), and work experience (years). The psychological variables were burnout, perceived stress, job satisfaction, coping strategies, and general health.Burnout syndrome was measured using the Maslach Burnout Syndrome (MBI)  adapted Perceived stress was measured using the Perceived Stress Scale  adapted The questionnaire used to assess job satisfaction was the Font–Roja questionnaire . It consThe Spanish version  of the BHealth status was measured using the General Health Questionnaire (GHQ)  validateData research is available as The study was approved by the clinical research ethics committee . A cover letter explaining the voluntary and confidential nature of the study was delivered to all ED healthcare personnel.t-, Mann–Whitney, Analysis of variance and Kruskal–Wallis tests were used to analyze the relationships between the sociodemographic, occupational, and psychological characteristics and the MBI dimensions. Correlations between the quantitative variables were tested using the Pearson correlation and the Spearman’s Rho tests. Three univariate linear regressions were created in order to assess the relationship between the sociodemographic, occupational, and psychological characteristics and each MBI dimension. Those variables that showed a statistically significant relationship with each of the considered dimensions (p < 0.05) were subsequently included in a multivariate linear regression model. In this way, 3 elimination multiple linear regression models were created for each MBI dimension . For purposes of the multivariate analysis, the variables were reorganized as follows: marital status  and type of employment contract . All the results were considered statistically significant with the p-value < 0.05. The statistical analyses were performed with statistical package G-Stat V.2.0 .A descriptive analysis was performed using the means and the standard deviations for the quantitative variables, and frequencies and percentages for the categorical variables. The Kolmogorov–Smirnov test was used to check the normality of the variables. Student’s A total of 171 nurses participated in the study. The participants’ mean age was 47.85 (8.11) years, and 73.10% were women. Of the group, 60.23% were single, 48.54% did daily physical exercise, and 67.25% had a permanent contract. Other sociodemographic and work characteristics are shown in As regards the levels of burnout , ED nursp = 0.001) and social dysfunction (p = 0.002). A significant negative correlation was also found between perceived stress and job satisfaction (p = 0.0004). Burnout variables EE and DP and the subscales corresponding to the GHQ showed positive relationships with avoidance coping . Problem-focused coping was negatively correlated with depression (p = 0.003) and social dysfunction (p = 0.0002). It should also be noted that a significant positive correlation was found between emotion-focused coping and depression (p = 0.002).As shown in p = 0.005) scores. There were negative significant relationships between PA and age (p = 0.03), time of service at the ED (p = 0.03), and work experience (p = 0.02)  scores were evaluated according to the participants’ sociodemographic data and occupational characteristics. Accordingly, those who did not take part in daily physical exercise had higher mean DP ( = 0.02) .p = 0.03), anxiety (p = 0.02), social dysfunction (p = 0.02), and being female (p = 0.01) were statistically significant predictors of EE. DP was determined by the absence of daily physical exercise (p = 0.006), being female (p = 0.01), and the use of avoidance coping (p = 0.03). PA seems to be influenced by the years worked at EDs (p = 0.03).In this study, 21.05% and 43.27% of the participants had high levels of EE and DP, respectively. 26.31% had low PA. Among the nursing staff, the prevalence of each of the 3 dimensions of burnout according to the MBI range was as follows: high level of EE (20–44%), high level of DP (23–51%), and low level of PA (15–44%) . RegardiThe participants’ perceived stress score was within the normal range, similar to the results of Mirhagi and Sarabien . HoweverDespite the fact that the impact of working in an ED on the level of stress and burnout among nurses has now been established ,55,56, lIn the current study, somatic symptoms and social dysfunction were the most frequent clinical manifestations among ED nurses. This is consistent with the results of another study carried out among emergency and intensive nursing staff . The useWe found that EE and DP were influenced positively by being a female. However, this result should be viewed with caution due to the sample of our study consisting predominantly of women. While previous studies noted that gender is an important variable in EE and that women experience more burnout than men ,76, therIn this study, no relationship was found between marital status and the dimensions of burnout. In this, the findings from the literature are again unclear and contradictory. Some authors suggest that burnout is associated with people who have no partner , while oThe role of lifestyles in ED healthcare professionals’ burnout levels needs to be studied extensively . FurtherAs regards job characteristics, the correlational analysis showed an inverse relationship between work experience and PA. In the multivariate analysis, PA was influenced negatively by the years worked in EDs. Working in an ED involves dealing with unexpected situations, patients who have life-threatening pathologies, and more frequent attacks or assaults than other specialized medical units, which may produce lack of assertive skills in nursing professionals and, as a result, low PA ,87. HoweNo differences were found between the type of employment contract and the dimensions of burnout, which was not consistent with the results from Garcia et al. , who revOur study has certain limitations. First of all, because of the cross-sectional study design, it is not possible to establish any cause-effect relationships. Secondly, findings may not necessarily be representative, as a convenience sample was used. Thirdly, the study was carried out only in the region of Andalusia, which may limit the generalization of the results. In further studies, it would be interesting to consider using a wider geographical range, and to use longitudinal research methods and randomized sampling.It is vital for health services to be aware of the relationships between burnout, perceived stress, job satisfaction, coping strategies, and general health. Since ED nursing professionals provide a valuable service to the community, the levels of these factors should be taken into account, as they have an important impact on patients, as well as on the general population. Understanding the influence that work characteristics have on burnout is crucial to inform policy and practice in designing suitable interventions to prevent illnesses and improve motivation among ED nurses.High burnout affects 8.19% nurses working in the EDs of four hospitals in the region of Andalusia. Perceived stress is within the normal range and job satisfaction level is moderate. Problem-focused coping is the most commonly used strategy, and somatic symptoms and social dysfunction are the most frequently experienced clinical manifestations. The absence of physical exercise, gender, years worked in EDs, anxiety, social dysfunction, and avoidance coping are the main predictors of burnout.


Objectives: This study compared neurofilament light chain (NFL) levels in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (sALS) with levels in patients with other neurological diseases and healthy controls and assessed correlations between NFL levels and clinical indicators of sALS.Methods: We used enzyme-linked immunosorbent assays to determine the NFL levels in the CSF of 45 patients with sALS, 21 patients with other central nervous system diseases (OCNSDs), 18 with immune-mediated peripheral neuropathy (IMPN), 14 with non-immune-mediated peripheral neuropathy (NIMPN), and 19 healthy controls (HCs).Results: The median NFL levels in the CSF of the sALS, OCNSD, IMPN, NIMPN, and HC groups were 6510, 5372, 4320, 1477, and 756 pg/mL, respectively. The CSF NFL levels did not differ significantly among the sALS, IMPN, and OCNSD groups, but were significantly higher than those of the NIMPN and HC groups. The NFL CSF levels were significantly higher in the NIMPN group than the HCs. There was a negative correlation between the NFL level and ALS function score (ALSFRS-R), and a positive correlation with the disease progression rate in patients with sALS.Conclusion: CSF NFL may not be sufficient to distinguish ALS from other central nervous system diseases or peripheral neuropathy, but it predicts ALS severity and progression. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively involves the upper and lower motor neurons. The pathogenesis of ALS remains unclear, but axonal injury may be involved . The diaThe study enrolled 45 patients diagnosed with sALS admitted to the Department of Neurology at the Chinese PLA General Hospital from December 2017 to December 2018. The diagnosis was based on the Revised E1 Escorial diagnostic criteria . Three dThe clinical data of the 45 patients with sALS were recorded, including age, gender, site of symptom onset, disease duration (time from symptom onset to sample collection), diagnostic category , and the time of symptom spread from spinal or bulbar localization to generalization (TTG), which is an early clinical parameter of disease progression , as wellCSF samples were collected from all patients and controls at the time of diagnosis in our department after lumbar puncture for CSF analysis. All samples were centrifuged at 3,000 rpm at 4°C for 10 min within 2 h of collection. The supernatants were stored at −80°C until tested. This study was approved by the ethics committee of the Chinese PLA General Hospital. Informed consent was obtained from all participants.The NFL CSF concentrations were measured using commercial enzyme-linked immunosorbent assays , performed in accordance with the manufacturer's instructions. The range 100–10,000 ng/L was explored. Samples were run in duplicate, together with freshly prepared standards, and positive and negative controls on each plate.p < 0.05.For continuous variables, the Kolmogorov–Smirnov test was used to determine whether the data were normally distributed and Levene's test to determine the homogeneity of the variance. Normally distributed data are expressed as the mean ± standard deviation and non-normally distributed data as the median and interquartile range (IQR). The CSF NFL levels were non-normally distributed. Differences in the CSF NFL levels between the HCs and sALS or OCNSD groups were determined by analysis of covariance adjusted for age. Wilcoxon's non-parametric test was used to compare the NFL levels among the other groups. Spearman correlation analysis was used to analyze the associations between the CSF NFL level and clinical parameters in the sALS patients. The analyses were performed using SPSS . The level of statistical significance was set at r = 0.339, p < 0.0001), but not with gender . Among the five groups, significant differences were found in age between the HCs and sALS (p = 0.016) and OCNSD (p = 0.010) groups. To avoid age-related bias when comparing NFL levels, differences in CSF NFL levels between the HCs and sALS and OCNSD groups were analyzed using analysis of covariance after adjusting for age . Of these, 28 were diagnosed with clinically definite ALS, 15 with clinically probable ALS, and two with clinically possible ALS. Eight cases were classified as bulbar onset and 37 as limb onset ALS. The mean age at sampling was 54.29 ± 8.81 years. The median disease duration was 12 (range 3–84) months. There were 37 patients with classical ALS and four each with UMND-ALS and LMND-ALS. The mean ALSFRS-r score was 37.29 ± 8.54 and the mean DPR was 0.94 ± 0.85. The mean FVC%, which was obtained from 39 ALS patients, was 87.23 ± 21.17%. Twenty-two cases progressed to generalized ALS and the mean TTG was 12.18 ± 11.30 months.The median CSF NFL levels in the sALS, OCNSD, IMPN, NIMPN, and HC groups were 6510, 5307, 4320, 1477, and 756 pg/mL, respectively . No signUsing Spearman correlation analysis, there were no significant correlations between NFL level and age at onset, onset site, diagnostic category, clinical phenotype, TTG, or FVC. A significant negative correlation was found between NFL level and ALSFRS-r and a significant positive correlation between NFL level and DPR , 3.In this prospective study, the CSF NFL levels were significantly higher in patients with sALS than healthy controls, but were not significantly elevated compared with patients with OCNSD or IMPN, which are expected to lead to significant axon damage. We confirmed that NFL levels are promising prognostic biomarkers for monitoring disease severity and disease progression of sALS.In recent years, there has been strong evidence from independent groups, large cohorts of patients, and multicenter studies that neurofilament light chains are potentially valuable ALS biomarkers , 21, 23.We found a positive correlation between CSF NFL levels and the rate of disease progression and a significant negative correlation between CSF NFL levels and ALS function scores. The rate of disease progression based on the ALS function score is an important indicator of the clinical progression and deterioration of ALS patients . Our worThere were some shortcomings to our work. First, the sample size is relatively small; a large sample size may be more convincing. Second, we assayed only the CSF NFL concentration, and not that in blood. A comparison of blood and CSF NFL levels in patients may better explain the results. In addition, serum measurements are more favorable to monitor disease progression, and the reliability of NFL assay also in serum was widely reported in literature. Third, all patients in our study underwent therapies (Riluzole or Edaravone) at the time of lumbar puncture. We did not record the detailed treatment process, which possibly influenced axonal preservation or damage, and future study should consider it.In conclusion, the CSF NFL levels are increased in patients with sALS and other nervous system diseases. The high CSF NFL levels of sALS patients were associated with more rapidly evolving disease and more severe disability due to ALS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of the Chinese PLA General Hospital. The patients/participants provided their written informed consent to participate in this study.QS was responsible for the design of the subject, the implementation of the experiment, and the writing of the article. XZ was responsible for the collection of sample. SL was responsible for the guidance of the experiment. FY, HW, and FC were responsible for the guidance of the subject. XH was in charge of the project, responsible for the project's funding, design, and checking of the article.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.


Encephalitis is a common central nervous system inflammatory disease that seriously endangers human health owing to the lack of effective diagnostic methods, which leads to a high rate of misdiagnosis and mortality. Glutamate is implicated closely in microglial activation, and activated microglia are key players in encephalitis. Hence, using glutamate chemical exchange saturation transfer (GluCEST) imaging for the early diagnosis of encephalitis holds promise.Staphylococcus aureus infection were used for preclinical research of GluCEST imaging in a 7.0-Tesla scanner. For the clinical study, six patients with encephalitis, six patients with lacunar infarction, and six healthy volunteers underwent GluCEST imaging in a 3.0-Tesla scanner.The sensitivity of GluCEST imaging with different concentrations of glutamate and other major metabolites in the brain was validated in phantoms. Twenty-seven Sprague–Dawley (SD) rats with encephalitis induced by p = 0.000).The number of amine protons on glutamate that had a chemical shift of 3.0 ppm away from bulk water and the signal intensity of GluCEST were concentration-dependent. Under physiological conditions, glutamate is the main contributor to the GluCEST signal. Compared with normal tissue, in both rats and patients with encephalitis, the encephalitis areas demonstrated a hyper-intense GluCEST signal, while the lacunar infarction had a decreased GluCEST signal intensity. After intravenous immunoglobulin therapy, patients with encephalitis lesions showed a decrease in GluCEST signal, and the results were significantly different from the pre-treatment signal (1.34 ± 0.31 vs 5.0 ± 0.27%, respectively; in vivo imaging biomarker for the early diagnosis of encephalitis. GluCEST will provide new insight into encephalitis and help improve the differential diagnosis of brain disorders.Glutamate plays a role in encephalitis, and the GluCEST imaging signal has potential as an  Encephalitis is serious inflammatory disease of the central nervous system (CNS) that is caused by many physiological or pathological factors . Accordiin vivo, which has also limited its applicability despite the fact that it can provide unparalleled opportunities for understanding diseases in terms of metabolism information , with the advantages of high soft tissue resolution and the non-invasive nature of the procedure, has been widely used to diagnose encephalitis in the clinic; however, it can only provide anatomical information and the physiological or biochemical information that can be acquired is limited . Moreoveormation . In addiormation . TherefoChemical exchange saturation transfer (CEST) is a relatively novel MR molecular imaging approach that utilizes a frequency selective radiofrequency (RF) irradiation pulse on particular exchangeable protons , thus resulting in attenuated water signals that can be measured via the loss of water signal intensity to indirectly characterize the microenvironment of the solution . CEST MRIn this study, we hypothesized that Glu is involved in the occurrence of encephalitis and may serve as a potential biomarker for the diagnosis of incipient encephalitis. Moreover, we hypothesized that GluCEST imaging could be used to predict encephalitis progression or prognosis. Our study may provide new insight into encephalitis and help improve the differential diagnosis of brain disorders.N-acetylaspartate , myo-inositol , creatine , glutamine , Glu (10 mM), and γ-aminobutyric acid ] were also used in our study . Before the CEST imaging scan, the nuclear magnetic resonance tubes were inserted into a phantom holder filled with 3% agarose gel to minimize susceptibility inhomogeneity.Glu  phantoms were first prepared for the optimization of GluCEST MRI parameters. To evaluate whether the CEST effect of Glu was concentration-dependent, different concentrations of Glu  were prepared and their pH was titrated to 7.0. To simulate the effect of other metabolites on GluCEST under physiological conditions, different concentrations of metabolites  were used in our study. The results show that Glu is the main contributor to the GluCEST signal, GABA and Cr provide a small contribution to the GluCEST effect, while the contribution of other metabolites  to the GluCEST effect was negligible  in the encephalitis areas was elevated 3 days after infection with S. aureus compared with the healthy control (HC) group , indicating that Glu was involved in the occurrence of encephalitis and that GluCEST imaging could be used for the diagnosis of encephalitis    ; 26.66 ± 3 days) ].F = 1.737, p = 0.2097), sex , or education . Similarly, no statistically significant difference was found in EEG  or disease duration  between patients with encephalitis and LI. However, the CSF WBC count in the encephalitis group was higher than that in the LI group , indicating that suspected encephalitis patients could be diagnosed with encephalitis without requiring biopsy. The detailed demographic characteristics of subjects are presented in All subjects successfully completed the experiment. No significant differences were noted among the three groups  in terms of age  (As shown in ntensity . Moreove= 0.000) . Z-spect= 0.000) .t = 20.205, p = 0.000), and the range of the signal intensity of the lesions was also reduced in the anatomical images (T2W imaging and T2 Flair)  . Howeverin vivo application of GluCEST imaging in both a rat model of S. aureus-induced encephalitis and in patients with encephalitis, as well as quantified GluCEST signals and evaluated treatment responses. GluCEST maps indicated higher glutamate concentrations in the encephalitis lesions compared with the control group. To date, several studies have demonstrated that Glu serves as the predominant excitatory neurotransmitter in the brain and is involved in learning, memory, emotion, cognition, and energy metabolism  in S. aureus-induced brain lesions was significantly higher than that in healthy controls (S. aureus and Clostridium novyi-NT (C. novyi-NT), respectively −S(3ppm)/S(−3ppm) to calculate the GluCEST signal. MT asymmetry is dependent on the applied saturation pulse parameters, and a strong saturation pulse (saturation power >3 μT) with short duration can largely mitigate the confounding effects of MT asymmetry. Thus, in our study, a strong saturation pulse with short saturation times was applied both in the phantom  and in vivo  models. In addition, a strong saturated power can reduce the interference of NOE effect. Although several post-processing techniques have been developed to increase the specificity by removing DS, semi-solid MT, NOE effect, and T1w influences, it is still challenging to remove overlapping CEST signals from other potential contaminations. For instance, proteins also have amines of approximately 3 ppm which could contaminate the GluCEST effect. Recently, Zhang et al. performed both in vitro and in vivo experiments and demonstrated that exchange-dependent relaxation rate (Rex) technology may be a good choice to address this problem , semi-solid non-specific MT, nuclear overhauser enhancement (NOE) effect, water longitudinal relaxation time (T1w), and nearby overlapping CEST signal influences. Variation of these parameters may cause misinterpretation of the GluCEST signal in encephalitis. Thus, correction of these non-specific factors is necessary. Since the DS effects are symmetric with respect to the water resonance frequency, they can be removed by asymmetry analysis; thus, we used the GluCEST formula S(−3ppm)−S(3ppm)/S problem , 2018. IDespite the fact that our study confirmed that Glu was involved in the occurrence and development of encephalitis and can be quantitatively evaluated by GluCEST imaging, there are still some limitations in the present study. For example, (i) biopsy as the “gold standard” for the diagnosis of encephalitis, owing to its invasive nature and deep location, and the small scope of lesions limited its used in this study; (ii) the clinical sample sizes are small, and there was no classification of the encephalitis; and (iii) GluCEST imaging was affected by many factors, and there is a lack of unified clinical scanning and diagnostic standards, etc. In spite of these shortcomings, we are also working hard to solve them. For example, we tried our best to achieve accurate diagnosis through combined clinical symptoms, laboratory tests, and experienced neurologists. In terms of the insufficient sample size, we simulated the change in clinical encephalitis using the encephalitis rat model. Of course, we need to continue recruiting patients with encephalitis, and a grading study is also required. Finally, with the development of the MR system hardware and software as well as the efforts of scientists, a standard imaging scheme for clinical practice research will be developed in the future.in vivo imaging biomarker for metabolic and functional changes in the rat model of encephalitis induced by S. aureus and patients with encephalitis and could predict encephalitis progress or prognosis with highly clinically translatable value. Importantly, this may provide us new insight into encephalitis and help improve the differential diagnosis of brain disorders. In the future, it will be promising to develop novel agents based on Glu for targeted diagnosis and treatment of encephalitis.Our study demonstrates that Glu is associated with the early changes in the occurrence and development of encephalitis both in preclinical and clinical applications, and high-resolution GluCEST imaging could be used for real-time, quantitative, and non-invasive monitoring of its dynamic change without requiring the injection of any exogenous imaging contrast agents. In addition, we could use GluCEST imaging to observe the spatial alteration of Glu before and after treatment and then evaluate the therapeutic effect. Therefore, GluCEST imaging has the potential as an All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by the local ethics committee of the Second Affiliated Hospital of Shantou University Medical College. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Care and Use Committee of Shantou University Medical College.YChen and RWu designed the study. YChen, KG, YCheng, JQ, and HH performed the experiments and collected clinical data. YL and RWa processed images. YJ carried out statistical analysis and drafted the manuscript. YZ and RWu critically reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: To explore the association between ultrasound parameters and previous ischemic or hemorrhagic stroke in patients with moyamoya disease (MMD), and develop an ultrasound-based nomogram to identify stroke in patients with MMD.Methods: We prospectively enrolled 52 consecutive patients (92 hemispheres) with MMD at the Beijing Tiantan Hospital. Thirty-six patients (65 hemispheres) were assigned to the training dataset from September 2019 to February 2020, and 16 patients (27 hemispheres) were assigned to the validation dataset from March 2020 to July 2020. Multivariate logistic regression analysis was applied to identify ultrasound parameters associated with previous history of ipsilateral stroke in patients with MMD, and a nomogram was subsequently constructed to identify stroke in patients with MMD. The performance of the nomogram was evaluated with respect to discrimination, calibration, and clinical usefulness.Results: Multivariate analysis indicated that the flow volume (FV) of the extracranial internal carotid artery (EICA) and the peak systolic velocity (PSV) of the posterior cerebral artery (PCA) were independently associated with ipsilateral stroke in patients with MMD, a nomogram incorporating these two parameters was constructed to identify stroke patients. The area under the receiver operating characteristic (AUROC) curves was 0.776  in the training dataset and 0.753  in the validation dataset suggested that the model had good discrimination ability. The calibration plot showed good agreement in both the two datasets. The decision curve analysis (DCA) revealed that the nomogram was clinically useful.Conclusions: Ultrasound parameters of EICA and PCA are independently associated with history of previous ipsilateral ischemic or hemorrhagic stroke in patients with MMD. The present ultrasound-based nomogram might provide information to identify MMD patients with high risk of stroke. Future long-term follow-up studies are needed to prove the predictive value in other independent cohorts.Clinical Trial Registration:http://www.chictr.org.cn/index.aspx. Unique Identifier: ChiCTR1900026075. Moyamoya disease (MMD), which is a rare disease with unknown etiology, predisposes some patients to stroke. It is characterized by progressive stenosis of the terminal portions of the internal carotid arteries and their proximal branches. Reduced blood flow in the main vessels of the intracranial anterior circulation leads to compensatory development of abnormal vascular network , 2. LeptDigital subtraction angiography (DSA) is the traditional gold standard for pre-operative diagnosis and post-operative prognostic evaluation of MMD, nevertheless, there is concern regarding the invasiveness, high cost of the procedure and the associated exposure to radiation. In situations when DSA is not readily available, magnetic resonance angiography (MRA) combined with magnetic resonance imaging (MRI) is considered as an alternative tool , but MRAWe prospectively enrolled consecutive patients diagnosed with MMD based on the guidelines for MMD  between Informed consents were obtained from all eligible patients , and this study was approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University.Clinical information, including age, sex, clinical manifestations, radiological findings, history of hypertension, diabetes, hyperlipidemia, smoking, and drinking were recorded. Based on the clinical manifestations on admission, the 92 hemispheres from 52 patients were diagnosed as stroke or non-stroke by the following methods: (1) stroke was defined as patients who had already experienced ischemic stroke or hemorrhagic stroke. Ischemic stroke was a new symptomatic neurological deterioration that was confirmed by MRI as cerebral infarction and could not be attributed to non-ischemic causes. Hemorrhagic stroke was defined as blood permeated into the brain parenchyma that was confirmed by CT . (2) NonThe criteria of collateral circulation were defined by Liu et al. . AnterioPosterior collateral circulation was assessed according to the anatomical extent of pial collateral blood from the PCA territory to the middle cerebral artery (MCA) and anterior cerebral artery (ACA) territory . ScoringThe grading score of the collateral circulation was the sum of the above anterior and posterior collateral circulation scores, it was graded as follows: grade I, 0 to 4 points; grade II, 5 to 8 points; and grade III, 9 to12 points.Ultrasound parameters were measured by 2 independent experienced sonographers (SZ and YL), blinded to the radiographic findings and clinical data. Ultrasound examination was performed using a sonographic scanner .2 × π]  of the EICA was measured at 1–2 cm above the carotid sinus. Then color and pulse doppler were used to measure the time-averaged mean velocity (TAMV). The gain, pulse-repetition frequency, and wall filter were corrected to appropriate condition, the doppler sample size was adjusted to the same width as the vessel, the doppler angle was <60°, and after at least three cardiac cycles, the TAMV was measured. The flow volume (FV) was obtained according to the formula FV = TAMV × [(D/2)2 × π] , 14. TheThe STA was examined using a 3–12 MHz linear array probe. The patient assumed a supine position with the head turned to the opposite side. The probe was placed before the ear at the trunk of the STA. The examiner adjusted the depth, grayscale, and focus according to the condition of each patient. Color and pulse doppler were subsequently used to measure the peak systolic velocity (PSV) of the STA. The gain, pulse-repetition frequency, and wall filter were corrected to appropriate condition, the doppler sample size was adjusted to about 1/3 of the STA, and the doppler angle was <60°. Then, the PSV of the STA was measured. The level of the PSV in STA was categorized as “ <50 cm/s” or “50–99 cm/s.”The MA was examined with a 1–5 MHz convex transducer. The patient assumed a supine position with the head turned to the opposite side. The probe was placed at the mandibular angle and pointed toward the tip of the nose. The examiner adjusted the depth, grayscale, and focus according to the condition of each patient. Color and pulse doppler were subsequently used to measure the PSV of the MA. The gain, pulse-repetition frequency, and wall filter were corrected to appropriate condition, the doppler sample size was adjusted to about 1/3 of the MA, and the doppler angle was <60°. Then, the PSV of MA was measured. The level of the PSV in MA was categorized as “<50 cm/s,” “50–99 cm/s,” or “100–149 cm/s.”Transcranial color-coded sonography (TCCS) was performed using a 1.5–3.0 MHz phased array probe. The P2 segment of the PCA was detected through the transtemporal window. Color and pulse doppler were used to measure the PSV of the PCA. The patient assumed a lateral position. The examiner adjusted the parameters of the sonographic scanner according to the condition of each patient. The depth of insonation was adjusted to 60–70 mm, the doppler sample size was 3–5 mm, the doppler angle was <60°. Then, the PSV of the PCA was measured. The level of the PSV in PCA was categorized as “<50 cm/s,” “50–99 cm/s,” “100–149 cm/s,” “150–199 cm/s,” or “>200cm/s.”U-test was used for continuous variables and ranked variables; the chi-square test was used for categorical variables. Weighted kappa (κ) was used to assess the interrater reliability of ultrasound parameters between the 2 sonographers in the training dataset. As for ultrasound parameters we used the parameters measured by the first rater. Multivariate logistic regression analysis was applied to identify ultrasound parameters associated with previous history of ipsilateral stroke in patients with MMD. The rms package for R software 3.6.1 was used to establish a nomogram incorporating the independent associated parameters. Then, a validation dataset was used for verification. The discrimination ability of the nomogram was evaluated using the area under the receiver operating characteristic (AUROC) curves. The consistency between the observed and the assessed probability of stroke was evaluated by calibration curve. The Hosmer-Lemeshow-test was performed to verify the suitability of the nomogram . Continuous variables were described as medians (interquartile ranges), ranked variables and categorical variables were described as percentages. The Mann-Whitney nomogram , 16. MeaDuring the study period, of 205 patients with MMD, 13 patients refused ultrasound examination, 8 patients with poor temporal acoustic bone window, 10 patients with diseases that affect cardiac output, 48 patients underwent prior bypass surgery and 74 patients lack of DSA, CT, or MRI examination, or the interval between these examinations and ultrasound was more than 1 month, or during this period, patients exhibited new symptoms or clinical manifestations of sudden aggravation were excluded. Finally, 52 patients (92 hemispheres) met the inclusion criteria were included in the analysis. Among them, from September 2019 to February 2020, 36 patients (65 hemispheres) were assigned to the training dataset, from March 2020 to July 2020, 16 patients (27 hemispheres) were assigned to the validation dataset. Of 52 eligible patients, 12 patients had unilateral hemispheric involvement and 40 patients had bilateral hemispheric involvement. The demographics, clinical history, angiographic findings and ultrasound parameters of patients with MMD in the training and validation datasets were shown in The interrater reliability between 2 sonographers was good for the FV of the EICA  and the PSV of the MA ; The interrater reliability was excellent for the PSV of the STA  and the PSV of the PCA .P = 0.005) and the PSV of the PCA  were independent associated ultrasound parameters for stroke. After adjusting for age and sex in model 2, the FV of the EICA  and the PSV of the PCA  were still independent associated ultrasound parameters for stroke  in the training dataset and 0.753  in the validation dataset suggested that the model had good discrimination ability , 4A. Thedatasets , 4B. Thedatasets .In this study, we constructed an easy-to-use nomogram incorporating the FV of the EICA and the PSV of the PCA to identify previous history of ipsilateral stroke in patients with MMD. The discrimination ability of the nomogram was evaluated by using the AUROC, with an AUROC of 0.5 being defined as meaningless; 0.5–0.7 being defined as fair; 0.7–0.9 being defined as good; and >0.9 being defined as excellent. Our nomogram showed good discrimination and good calibration in both the training and validation datasets. The DCA indicated that the nomogram was clinically useful.The clinical manifestations and outcomes of patients with MMD are heterogeneous. Digital subtraction angiography (DSA) is the traditional gold standard for pre-operative diagnosis and post-operative prognostic evaluation of MMD. According to DSA findings, the severity of the disease is often divided into six progressive stages, which is called as Suzuki stage and were defined by Suzuki and Takaku in 1969 (1). However, DSA is invasive and requires exogenous contrast agent and ionizing radiation. Although it has improved our understanding of MMD, it is not an ideal method for long-term monitoring and assessing risk of poor clinical outcomes in patients with MMD.Ultrasound is a non-invasive, repeatable, and economical technique, that has been used to screen MMD, detect moyamoya spontaneous anastomosis pre-operatively and evaluate the prognosis post-operatively –10. HongHowever, in some cases, the Suzuki stage may not correlate with clinical severity, because the main blood vessels of anterior circulation occlusions may be compensated by collateral vessels. Leptomeningeal collaterals from the PCA are regarded as the main collateral vessels, and transdural collaterals from the ECA can also compensate for ischemia in the brain , 22, 23.Recently, Liu et al. proposed a new MMD grading system to assess clinical symptoms by combining Suzuki stage with the leptomeningeal system from the PCA. This new MMD grading system correlates well with hemodynamic status and clinical symptoms, and contributes to risk stratification and prognostic predictions in patients with MMD . In our Our study has some limitations. First, because the incidence of MMD is low, our sample size was relatively small. We divided the patients into stroke group and non-stroke group according to their clinical manifestations, but did not further divide the patients into ischemic stroke group, hemorrhagic stroke group, transient ischemic attack group, and asymptomatic group. Second, this study was a single-center study, and there was no ultrasound examination before stroke onset in patients with MMD. Therefore, Further validation with prospective registration and long-term follow-up is needed to prove the predictive ability of our findings.Ultrasound parameters of EICA and PCA are associated with previous history of ipsilateral stroke in patients with MMD. The present ultrasound-based nomogram may provide information to identify MMD patients with high risk of stroke, large-scale and prospective longitudinal cohort studies are needed to validate its predictability.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of Beijing Tiantan Hospital, Capital Medical University, Beijing, China. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.SZ, PG, YL, and JW: conception and design. SZ, PG, YL, JW, ZS, JZ, and LH: acquisition of data. SZ and PG: analysis and interpretation of data. SZ: drafting the article. WH: approved the final version of the manuscript on behalf of all authors. LC, DZ, and WH: study supervision. All authors critically revising the article and reviewed submitted version of 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.


Histidine is an essential amino acid (EAA) in mammals, fish, and poultry. We aim to give an overview of the metabolism and physiological effects of histidine in humans and different animal species through a systematic review following the guidelines of PRISMA . In humans, dietary histidine may be associated with factors that improve metabolic syndrome and has an effect on ion absorption. In rats, histidine supplementation increases food intake. It also provides neuroprotection at an early stage and could protect against epileptic seizures. In chickens, histidine is particularly important as a limiting factor for carnosine synthesis, which has strong anti-oxidant effects. In fish, dietary histidine may be one of the most important factors in preventing cataracts. In ruminants, histidine is a limiting factor for milk protein synthesis and could be the first limiting AA for growth. In excess, histidine supplementation can be responsible for eating and memory disorders in humans and can induce growth retardation and metabolic dysfunction in most species. To conclude, the requirements for histidine, like for other EAA, have been derived from growth and AA composition in tissues and also have specific metabolic roles depending on species and dietary levels. Histidine is an essential amino acid in mammals, fish and poultry because it is not de novo synthesized and must be obtained through the diet ,2. In thDepending on the species, the requirements and toxicity of histidine differ, as does its effect on metabolism. Therefore, our aim is to present a systematic review following the guidelines of PRISMA  methodo of the mHistidine requirements are expressed differently among species. For humans, histidine requirements are expressed as daily intake per kilogram body weight. For other species, lysine is frequently used as the reference AA, being known as the first limiting AA. Hence, the requirement patterns for other AAs, including histidine, are expressed in ratio to lysine requirements. According to the Food and Agriculture Organization of the United Nations (FAO), the histidine ratio (percent histidine/lysine) varies: 36 for pigs (20 to 50 kg), 32 for chicks (0 to 3 wk), 36 for rats, 33 for salmonids, 39 for trout, 37 for common carp and 34 for tilapia.Histidine undergoes different metabolic pathways . It can 5-formimino-tetrahydro-folate. In epiderma, urocanate hydratase is not present, and urocanic acid is the last compound of histidine catabolism. This compound accumulates in the epidermis and acts as a natural sunscreen that protects the skin from the effects of UV light [Urocanic acid has a protective role against harmful effects of ultraviolet rays on the skin. It is metabolized in the liver to 4, 5-dihydri-4-oxo-5-imidazolepropanoic acid by urocanate hydratase, and is then converted to formimino-glutamate (FIGLU) and NUV light ,9. HistiUV light . Its actUV light . Some caUV light . Gene exUV light . HistidaUV light . In the UV light . It was UV light . Lastly,UV light .Incorporation into protein: Borsook and Wolf reported that the main region for histidine retention was the viscera. This was determined by the injection of labeled AAs into rodents and the subsequent analysis of the rate of incorporation into protein tissue ,17. The Synthesis of carnosine and anserine: Carnosine is highly concentrated in muscle (except in cardiac muscle) and brain tissues of all mammals and birds, with a particularly high content in the horse . AnserinDecarboxylation of histidine to histamine by histidine decarboxylase: This metabolic conversion is common in all animal species except fish. This enzyme is present in microorganisms of the large intestine and in many tissues . In contrast to histamine, histidine can cross the blood brain barrier. After crossing the blood-brain barrier, and once in the tuberomammilary nucleus (TMN) of the hypothalamus, histidine is converted into histamine by histidine decarboxylase . SubsequHistamine is a neurotransmitter  that plaBuffering role of histidine and histidine-related compounds (HRC): Histidine and HRC have a specific role in the intracellular, non-bicarbonate buffering capacity of vertebrate muscle, which is mainly supported by the imidazole groups of free histidine or histidine residues in proteins as well as HRC, in the pH range of 6.5–7.5 . This meThe imidazole sidechain of histidine is a common coordinating ligand in metalloproteins. Histidyl residues of metal-activated enzymes and metalloproteins are believed to play an important role in metal binding by these proteins . HistidiAccording to the FAO, the daily requirement for histidine is 8 to 12 mg/kg of body weight per day in adults. The average intake of histidine in typical, adult diets in Europe, the USA and Japan was reported to be between 2.12 and 2.40 g per day , where men of 50–70 years of age at 5.20 g/day represented the 99th percentile intake ,33.Steele and Le Bovit  have stup < 0.05).Henkin  demonstrIn order to investigate the possible efficacy of oral histidine as anorectic therapy in men, Schechter and Prakash  fed eighGeliebter et al. studied the effect of daily histidine doses, from 24 to 64 g, mixed into orange juice, on healthy subjects for four weeks . They reSome studies have investigated the effect of histidine on ion absorption. In 113 subjects, for example, iron absorption was not affected by histidine supplementation (416 to 2080 mg/day) . In anotATP7A gene, and can lead to death in early childhood. Symptoms of this disease are attributed to deficient activity of Cu-dependent enzymes . In patients suffering from MD, copper therapy has been shown to be inefficient. Interestingly, however, due to the chelating effect, copper histidine therapy is beneficial in reversing the skin and hair changes, improving appendicular tone, socio-cognitive milestones, weight gain, and immunity [Menkes disease (MD) is a neurodegenerative disorder caused by mutations in the immunity ,45,46.Histidine status seems to be important in skin dysfunction and various cutaneous diseases. The deficiency in histidine, as in other essential AA  significantly decreased hyaluronan levels in human dermal fibroblasts . HyaluroDietary histidine may be associated with factors improving metabolic syndrome related to obesity. An internet-based, cross-sectional study conducted in a northern Chinese population by Yan-Chuan Li and colleagues  reportedObesity is also associated with a high level of pro-inflammatory cytokines and inflammation biomarkers . In the Many studies have shown that histidine acts on feeding behavior . ResultsIn contrast to these studies showing a decrease in food intake, a study by Holeček demonstrated an increase in food intake in rats that drank histidine-enriched water . They hyCerebral ischemia is a major cause of death in adults. Six hours after a cerebral ischemia, astrocytes are activated to induce neuron survival . To avoiAn early study by Liao and colleagues  showed tSome studies have reported that histidine could protect against epileptic seizures. A study by Chen et al.  showed tIn a study in which the level of histidine was >2 g/kg BW/day, rats from 7 to 46 weeks showed growth retardation, hepatomegaly and hypercholesterolemia ,67; whenAn excess of histidine can have a negative impact on growth, but the effect can be mitigated by increasing dietary protein content. Muramatsu et al.  studied 59Fe solution that contained ascorbic acid, it increased absorption [65Zn transport across the brain endothelium when histidine was added to perfusion [65Zn from two to four weeks after a single injection of the tracer), whereas the diet supplemented with 8% histidine induced a severe zinc deficiency (50% reduction in the plasma zinc content) [65Zn tissue distribution, and tissue-specific activities). However, when zinc intake was low, histidine supplementation led to a lower 65Zn retention, associated with increased fecal excretion and a shorter biological half-life [As previously described, histidine can chelate metal ions. Some studies have investigated the effect of histidine intake on iron, copper and zinc absorption and retention. Tissue levels of iron and fecal losses of iron were not affected by the histidine supplementation . When hisorption . This susorption . In vitrsorption ,78,79,80sorption  investigerfusion . Snedekeerfusion  reportedcontent) . This efalf-life .HAL), and amidohydrolase domain containing 1 (AMDHD1) decreased the sensitivity of cells to methotrexate. Moreover, methotrexate and histidine treatment of mice induced a marked decrease in tumor size that was significantly greater than in any of the other treatment cohorts. Taken together, these data highlight the role of the histidine degradation pathway in the efficiency of chemotherapy agent methotrexate [Recent data suggest that histidine catabolism and intake influence the sensitivity of cancer cells to methotrexate . Methotrotrexate .Toxicity doses have been studied in rats from different perspectives: their carcinogenicty as well as lethal doses. No mortality or adverse effects were observed during a 13-week period during which rats were fed with different amounts of histidine  . AnotherFigueroa and colleagues  studied Few studies have investigated the effect of histidine supplementation in chicken. High supplementation of histidine (4 g/kg on an as fed basis) was shown to reduce average daily gain by 48 to 51% in eight-day-old crossbred chicks . SimilarKopec studied the effects of histidine and zinc supplementation on broiler chickens . To do sIn conclusion, the addition of histidine in the diet can increase carnosine and anserine and improve meat quality.In grass carp fed a histidine-supplemented diet for two weeks , the dietary histidine requirement of young grass carp (279.1–685.4 g) was 7.63 g/kg, based on growth performance . Above tIn catfish, the requirement was demonstrated to be 3.7 +/−0.1 g/kg of histidine based on dry diet, or 15.4 g/kg of histidine when expressed as a percentage of dietary protein. Dietary histidine caused no increase in serum-free histidine levels until the dietary requirement was reached. Muscle carnosine could not be detected in the catfish . In NileSalmo gairdneri) and the Pacific blue marlin (Makaira nigricans). They found that HRC contribution to total cellular buffering varied from a high of 62% for white marlin muscle to a low of 7% for red trout muscle. These results suggest that the HRC buffering capacity depends on fish species and type of muscle. In red muscle, other principal buffers are phosphate and protein with taurine. Buffering capacities of histidine could act through N -acetylhistidine (NAH) presence in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, but not in those of endothermic birds and mammals. Although NAH is known to be synthesized from histidine and acetyl-CoA by histidine N-acetyltransferase , the gene that encodes HISAT remains unidentified. It was observed that 13 species  contained considerable amounts of NAH in their skeletal muscles (>1 µmol/g). The highest level of NAH (10.37 µmol/g) was found in the tissue of Betta splendens (Siamese fighting fish). Moreover, the NAH contents in the tissues of Trichogaster trichopterus (three spot gourami), Kryptopterus bicirrhis (glass catfish), Oreochromis niloticus (Nile tilapia), Mikrogeophagus ramirezi (ram cichlid), and Parachromis managuensis (Guapote tiger) were 3.17–−6.16 micromol/g. The skeletal muscle of amphibians (five species) and reptiles (four species) had a low level (< 0.25 µmol/g) of NAH [Fish contain a large amount of free histidine and HRC. The HRC buffering capacity has been studied by Abe and al.  in two f) of NAH .Another functional aspect of histidine is its capacity to decrease the incidence and severity of cataracts in fish. Cataracts are a major concern in the aquaculture industry. This function of histidine was suggested after an outbreak of cataracts in rapid growth Atlantic salmon fed with feeds free of blood meal, a good source of histidine in the diet .N -acetylhistidine (NAH) is a prominent biomolecule in the lens; lens NAH concentrations directly reflect dietary histidine levels in Atlantic salmon. Dietary histidine appears to be one of the important factors in preventing cataracts. The beneficial effects are related to high levels of histidine and the buildup of NAH in the lens, which possess buffering and antioxidant properties . In cultSchoof et al.  exploredOn the other hand, a study by Gabel and Poppe  used a f2448. This study proved that there is another phosphorylation site, Ser2481, that is an indicator of mTOR pathway activation in CMEC-H. P-mTOR was increased with addition of histidine in the full concentration range . They also studied the effects of histidine on Raptor and GβL, two other mTORC1 components. Their expression was also increased with histidine supplementation compared to the control group. Concerning S6K1, an mTOR target, there was an increase in phosphorylation within all concentration ranges. This result contrasts to what was found by Prizant and Barash [Gao and colleagues studied the effects of leucine and histidine, separately, on the mTOR signaling pathway in milk protein synthesis . To do sd Barash , but is d Barash . In the M, while the concentration of other AAs was not affected. Moreover, the supplementation with histidine had an impact on milk yield with an increase of 1.7 L/d, and on lactose yield with an increase of 90 g/d. This was accompanied by an increase in protein yield and a decrease in fat percentage. However, the effect of water intake was not discussed.Another study investigated the effects of histidine-supplemented drinking water on the performance of lactating dairy cows . Their hS. Hadrova et al.  evaluateHistidine is an essential AA and its requirements have been derived from growth and AA composition in tissues. In addition to its role in protein metabolism, histidine, as a functional AA, has specific metabolic roles.In human, the daily requirement for histidine is 8 to 12 mg/kg of body weight per day in adults, and the average intake in typical adult diets in Europe, USA and Japan was reported to be between 30 to 35 mg/kg of body weight per day (2.12 and 2.40 g per day). Daily histidine supplementation, under 2 g/daily, improves inflammation in overweight and obese people, decreases feelings of fatigue, and increases concentration and efficiency at work. A daily intake of 4 g decreases severity in atopic dermatitis disease. If the supplementation increases up to 8 g of histidine, a severe anorexia is induced. Finally, supplementation with a large dose, from 16 to 64 g/day, induces a decrease in taste and smell acuity, and the onset of headaches, weakness, drowsiness, nausea, and memory disorder.In rodents, histidine supplementation up to 25 g/kg of diet is beneficial. A large dose of 50 g/kg of diet can have different adverse effects, such as a large decrease in body weight gain, in food intake, and a growth retardation. Due to this negative effect, maximal tolerable dose of histidine was fixed to 25 g/kg of the diet.In pigs, histidine, when combined with other amino acids, can improve performance and amino acid balance in a context of low-protein diet.In chickens, the major benefit of histidine supplementation is the increase in carnosine and anserine in the muscle allowing an increase in anti-oxidant capacity.In salmonids, a beneficial effect of histidine supplementation is its capacity to decrease the incidence and severity of cataracts.In ruminants, histidine supplementation induces an increase in milk yield and an increase in milk protein synthesis.As such, when histidine is limiting in the diet in relation to the need for growth, its supplementation can be associated with a better performance. When this supplementation provides histidine beyond growth requirement, other benefits can be observed as described exhaustively in this paper. However, when this supplementation provides histidine in excess, AA imbalance can trigger a decrease in growth and food intake and other unwanted effects. These dose-effects of histidine are summarized thereafter:To conclude, histidine supplementation can be interesting for different aspects depending on the species. In humans, data suggest both clinical and nutritional interests of histidine supplementation as a strategy to improve metabolic syndrome features, skin dysfunctions, and memory. In animal production, histidine supplementation can be of interest to promote both the yield and quality of end-products. The indispensability of histidine, and the accumulating evidence of its importance in numerous physiological functions make histidine a unique AA which deserves further consideration in both human and animal nutrition.


Backgrounds: We have aimed to establish nerve ultrasound reference data in 8 to 17-year-old children and adolescents and to compare those data to younger children, adults, and age-matched children with polyneuropathies.Methods: High-resolution ultrasounds of the nerves were performed in 117 healthy children and adolescents at 20 predefined landmarks in the neck and the extremities of both sides. Mean values, side-to-side differences and intraneural ratios, as well as upper limits have been calculated. In a second step, a comparison between 25 children and adolescents of the same age range with proven hereditary and acquired neuropathies and lysosomal storage diseases has been carried out.Results: Nerve growth correlates significantly with age and reaches adult values at the age of around 15 years. The influence of body mass index and gender is negligible at most segments. By the use of age-specific upper limits, nerve enlargement could be seen in distinct types of neuropathies, particularly in demyelinating hereditary and inflammatory types, which is comparable to findings in adults, but also in rare lysosomal storage diseases.Conclusion: Nerve size correlates with age during childhood and reaches a climax in younger adults. Age-matched reference data are inevitable to differ between hypertrophic and non-hypertrophic nerve damage, e.g., in neuropathies. Nerve imaging in nerve pathologies has recently become a main focal point. By the use of magnetic resonance neurography and high-resolution ultrasonography, more knowledge about nerve morphology has been established. In HR-US, nerve enlargement due to tumors, neuromas, or inflammation has been the most profound finding –4. Thus,Healthy children/adolescents of 8 to 17 years of age were recruited from schools in Tuebingen County between May 2018 and 2019. The study was approved by the local ethics committee (Tuebingen 765/2017BO1). The informed consent was explained to the probands and signed by a parental authority. A medical questionnaire containing medical history, developing mile stones, family medical history, height, weight, gender, ethnicity, and handedness was performed next, followed by a short neurological examination. Exclusion criteria were confounding diseases, such as known untreated diabetes mellitus.In a second step, ultrasound results of 25 children and adolescents of the same age range with a definite diagnosis of acquired or inherited polyneuropathy were analyzed. Statistical comparisons to the reference group were not carried out due to the small sample size of the groups. Informed consent was retrospectively obtained.Nerve ultrasounds were carried out by CS and ASG blinded to each other by the use of a high resolution linear transducer . The authors were orientated on protocols, and they had previously performed other studies involving adults , 11–14. In all nerves, the cross-sectional area was measured (CSA), except for the roots, which were measured in a long-axis view as diameter. The measurement was carried out just inside the hyperechoic rim of the nerve .Furthermore, we calculated the intranerve-CSA-variability  in thoseDescriptive statistics were used to describe the sample, and for some statistical analyses two groups were built (Group A children were aged between 8 and 12 years and group B adolescents between 13 and 17 years). Continuous variables were analyzed either as mean and standard deviation (±SD) or as median and interquartile range (IQR) depending on the distribution of the data. Normal distribution was assessed by investigating kurtosis, skewness, histograms, and Q-Q plots. Categorical variables were described as percentages and absolute frequencies. In addition, to determine the reference ranges of the assessed nerves, we calculated the 95% confidence intervals, defined as the interval between which 95% of the values of a reference population fall into.T-tests were used to compare numerical variables that were approximately normally distributed, whereas Mann-Whitney tests were used to evaluate non-normally distributed variables.A bivariate analysis was performed to compare baseline characteristics. A Chi square test for categorical variables was used. Independent-sample The intranerve variability was calculated as the ratio between maximum CSA and minimum CSA. The side-to-side difference was calculated as the difference between the right and left CSA. A dependent-sample t test was used to find out whether these variables were normally distributed, while a Wilcoxon Test was used to evaluate non-normally distributed variables. The percentage of the side-to-side difference was calculated as the ratio of this difference vs. the right CSA. The relationship between CSA with demographic parameters was evaluated using an analysis of covariance (ANCOVA), with age, BMI, and gender being the independent variables. Several assumptions were tested before the model was carried out: normal distribution of residual, homocedasticity, and homogeneity of variance. The intrarater and interrater intraclass correlation coefficient (ICC) was calculated through the use of post-processing.The percentile curves of the nerves were calculated by the LMS method using R's VGAM package version. The LMS method plots the shapes of the percentiles by three uncorrelated curves: the L, M, and S curves. The first defines the skewness (L) of the distribution at each age, the second the median (M), and the third the coefficient of variation (S). The three parameters are constrained to change smoothly as the covariate changes, and they can be plotted against the covariate , 16. Thep ≤ 0.05, and results are reported with 95% confidence intervals (95% CI). The analyses were carried out using SPSS  and R Software version 3.6.All statistical tests were two-tailed, the significance level was set at p = 0.519); furthermore, more children were left-handed in this group . As expected, the BMI in group B was higher than in group A (p < 0.001)  have been included. One girl had well-treated diabetes mellitus type 1 without secondary organ affection. In Group A, a non-significant slight female dominance occurred compared to Group B (001) see . All meap < 0.05). Boys showed higher CSA values in the median nerve and the proximal tibial nerve but not elsewhere (p < 0.05). This side preference, however, was not found in the case of the tibial nerve and root C5 (p > 0.05). The percentage of the side-to-side difference of the MN, the TN, and root C5 did not show significant differences between the age groups (p > 0.05), ranging between 1 and 5% each nerve in the mean (SD up to 30%). The intranerve–CSA variability was 1.36 (SD 0.38) in the MN, 1.25 in the UN (SD 0.34), and 3.23 (SD 1.11) in the TN. Between group A and B, no significant differences were found (p > 0.05).The nerve CSA correlated significantly with age in all measured segments see . Concernlsewhere . The rigUsing percentile calculation by the LMS method, we could define the percentile values for each age and each nerve segment . Figure At last, we compared our upper limits and normal values to children with distinct neuropathies. Overall, 25 patients aged between 8 and 18 years with distinct diagnoses have been included. Ultrasound examinations were carried out using the same device and mostly the same protocol, except for some segments in some patients. All children and adolescents with genetically proven Charcot Marie Tooth neuropathy (CMT) 1a (gene: PMP 22 duplication), CMT1b (MPZ), CMT4c (SH3TC2), and CMT4d (NDRG1), which are all hereditary neuropathies with demyelination; were also revealed to have enlarged nerves, the majority of them present in most segments compared to the age-matched reference values. In contrast, axonal variants, i.e., CMT2, showed no nerve enlargement. Further demyelinating neuropathies, in detail lysosomal storage diseases  also showed multifocal nerve enlargement. A patient with very rare inherited neuropathy  revealed also nerve enlargement, whereas those patients without neuropathy (one with diffuse pain syndrome and one with cognitive deficits) revealed normal nerve values. Inflammatory neuropathies showed multifocal proximal predominant nerve enlargement with involvement of upper arm segments in CIDP and root/vagus predominance in GBS. Two patients with neurofibromatosis type 1 have also been analyzed, of whom one showed multiple nerve enlargements due to nerve tumors, and the other showed overall normal segments due to missed tumors. Reference data are the basis for correct nerve ultrasound performance –8. With With regard to reference values of children <8 years , nerve gThus, according to the obvious age dependency in adolescent nerve growth, normal values for each age were defined  in contrWith regard to several published adult data, nerves in school children and adolescents are smaller until the age of 15 to 17 –9. At thIt remains uncertain why nerve growth reaches a peak in young people and then even decreases again in seniority; however, one explanation might be reduced fluidity, reduced elasticity, and increased stiffness in the nerves of older people . InteresIn a second step of this study, we compared our data to age-matched patients with a proven diagnosis of neuropathy as proof concept. As in adults , 4, chilFinally, some patients with very rare neuropathy types, e.g., lysosomal storage diseases  haven been examined. They even exhibited nerve enlargement, which might be a sign of intraneural accumulation of metabolites , 21. ThiMoreover, in two patients with neurofibromatosis (NF) type 1, we could differentiate between one child with high peripheral tumor load and another without any peripheral tumor; this might underline the importance of ultrasound screening in the surveillance of patients with peripheral nerve sheath tumors, as also for adults the finding of all (nerves)-or-nothing has been described for NF1 .th Percentile, which is a compromise solution between sensitivity and specificity. (5) Reference data for C5 and C6 in young children have to be interpreted with caution, as only a part of the participants was examined sufficiently at this part of the neck. We suggest that plexus parts, i.e., the trunks, might be easier to visualize, and, thus, further studies would be needed concerning the plexus in children. (6) We must confess that the number of lefthanded individuals was somewhat higher in the older group than in the younger; however, the overall influence of the handedness was not statistically significant in our study. (7) Overall conclusion concerning the ultrasound pattern in distinct neuropathies in children/adolescents is not possible out of these data due to the small sample sizes of the patient group. For the same reason, statistical analyses have not been carried out. Further studies concerning those entities are needed. (8) Echointensity and fascicle counting are of further interest in study ultrasounds; however, our device was not suitable enough to visualize those data in children and adolescents. Herein, ultrahigh-resolution probes might be helpful (The interpretation of our data must be done with care: (1) our data have been collected within Caucasian population and thus might be different in countries with predominantly other ethnicities; further reference studies are therefore needed. (2) Ultrasound devices might show differences concerning normal values. Each lab might therefore create their own normal data values, although we suggest the device-independent stability of CSA measurements to be high. (3) Our comparison between children <8 and >8, adolescents and adults has been carried out based on distinct study group data; nevertheless, the protocol and the device were the same as the standardized one, and examiners were blinded to each other. The interrater and the intrarater ICCs were excellent in all studies. (4) Our upper limits have been calculated by using the 90 helpful .Alltogether, these data are the first normal data for a large cohort of children >7 years and adolescents. For each age, normal values seemed to be necessary as growing correlates significantly with aging. The influence of gender and BMI in our cohort was negligible. Side-to-side differences at some nerve landmark (1 to 5% in mean) must be kept in mind before defining pathology. These reference values simplify the analysis of children and adolescents with neuropathic disorders and clarify underlying diagnoses in many cases. These data enable initiations of nerve imaging studies in distinct pediatric neuromuscular disorders.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Tuebingen University Ethic committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.A-SG, CS, AG, NW, and SG were involved in the project development, the data acquisition, the data analysis and the drafting of the manuscript. J-HS, HK, VH, JK, and LS-H were involved in data acquisition and analysis. All authors carefully read the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.