Several milk/whey derived peptides possess high in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. However, in some cases, poor correlation between the in vitro ACE inhibitory activity and the in vivo antihypertensive activity has been observed. The aim of this study is to gain insight into the structure-activity relationship of peptide sequences present in whey/milk protein hydrolysates with high ACE inhibitory activity, which could lead to a better understanding and prediction of their in vivo antihypertensive activity. The potential interactions between peptides produced from whey proteins, previously reported as high ACE inhibitors such as IPP, LIVTQ, IIAE, LVYPFP, and human ACE were assessed using a molecular docking approach. The results show that peptides IIAE, LIVTQ, and LVYPFP formed strong H bonds with the amino acids Gln 259, His 331, and Thr 358 in the active site of the human ACE. Interestingly, the same residues were found to form strong hydrogen bonds with the ACE inhibitory drug Sampatrilat. Furthermore, peptides IIAE and LVYPFP interacted with the amino acid residues Gln 259 and His 331, respectively, also in common with other ACE-inhibitory drugs such as Captopril, Lisinopril and Elanapril. Additionally, IIAE interacted with the amino acid residue Asp 140 in common with Lisinopril, and LIVTQ interacted with Ala 332 in common with both Lisinopril and Elanapril. The peptides produced naturally from whey by enzymatic hydrolysis interacted with residues of the human ACE in common with potent ACE-inhibitory drugs which suggests that these natural peptides may be potent ACE inhibitors. The number of people with unhealthy living habits who have developed cardiovascular disease (CVD) has increased in recent years. The WHO reported that an estimated 17.9 million people lose their lives as a result of cardiovascular disease every year . High blACE  is a zinc metallopeptidase, found in male genital, vascular endothelial, neuro-epithelial, and absorptive epithelial cells ,8,9, andConsequently, ACE-inhibiting natural products have been vigorously investigated during the last decades, due to their potential in lowering blood pressure during hypertension. Among various types of bioactive peptides, ACE-inhibitory peptides from food sources have been most extensively studied for their potential use as natural alternatives to drugs for reducing blood pressure through binding and inhibiting ACE, and thus preventing and managing hypertension ,15. FoodAlthough different animal and plant proteins have been used in the development of functional foods providing antihypertensive activity, milk is the main source of antihypertensive ACE-inhibitory peptides reported to date . Milk isThe activity of these peptides depends on their inherent amino acid composition and sequence ,30. ShorACE inhibitors are commonly discovered using classic investigation techniques which include hydrolysis of proteins with different proteolytic enzymes, isolation and purification of peptides using chromatographic systems, and synthesis of corresponding peptides for the confirmation of activity and structure . AlthougHerein we investigate the potential interactions between whey protein derived peptides with high ACE inhibitory activity and human ACE, utilising a molecular docking approach. This study follows our previous work , where tAccording to the EMBOSS NEEDLE results , there iMolecular docking was conducted to elucidate the potential molecular interactions between the whey-protein derived peptide sequences and specific amino acids at the binding site of human ACE. The peptide sequences were docked into the binding site of the human ACE, using the X ray crystallographic structure of the human ACE receptor (PDB code 6F9V). The extracted co-crystallized ligand, Sampatrilat,  was firsHydrogen bonds are a significant factor that contribute to the specificity and stability of protein-ligand interactions. Hydrogen bonds interactions were demonstrated to play a crucial role in stabilizing the docked ligand complexes . The disN-{1-[2-carboxy-3-(N-mesyllysylamino) propyl]-1-cyclopentylcarbonyl} tyr-osine) -r-osine)  is a potr-osine) . Overall, the docking results together with comparisons with the ACE inhibitory drugs provide strong evidence for the ACE inhibitory activity of IIAE, LIVTQ, and LVYPFP. In our previous work , IIAE, L50 is defined as the peptide concentration required to reduce the ACE activity by half. In our previous work where we characterized angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins , peptidehttps://www.ebi.ac.uk/) was queried for human and rabbit ACE amino acid sequences, together with known three-dimensional protein structures. Reviewed sequences were selected and the protein sequence files were downloaded. The accession codes for the human ACE and the rabbit ACE used in this work are P12821 and P12822, respectively. The two sequences were then uploaded to Emboss Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) for multiple sequence alignment and comparison.EMBL-EBI  , as provWhey protein-derived peptides Ile-Pro-Pro (IPP), Leu-Ile-Val-Thr-Gln (LIVTQ), Ile-Ile-Ala-Glu (IIAE), and Leu-Val-Tyr-Pro-Phe-Pro (LVYPFP) were used as ligands in separate docking runs. Docking was performed using the docking algorithm Surflex-Dock, as provided in Sybyl-X 2.1. The X-ray crystallographic structure of sampatrilat-Asp in complex with Angiotensin-I-converting enzyme  retrieved from the protein data bank (PDB) was chosen as the target protein for the docking studies, based on its high resolution structure co-crystallized with sampatrilat-Asp .The Biopolymer Structure Preparation Tool, with the implemented default settings provided in the SYBYL programme suite, was used to prepare the protein structure for docking; hydrogens were added to the protein structure in idealised geometries, backbone and sidechains were repaired, residues were protonated, sidechain amides and sidechain bumps were fixed, stage minimization was performed, and all water and any ligand molecules were removed. The three-dimensional (3D) structure of each ligand was constructed, using the “Build Protein” tool, as provided in Sybyl-X. Once constructed, charges were assigned to each atom of each molecule, using Merck Molecular Force Field (MMFF94) charges. Localized energy minimizations were then performed, and the final structure for each ligand in its lowest energy conformation was used for subsequent docking experiments. The resulting 3D coordinate files were converted to a MOL2 format for subsequent use in Surflex-Dock experiments, as provided in the SYBYL-X 2.0 software suite.Surflex-Dock is a search algorithm that utilizes an empirically derived scoring function whose parameters are based on protein-ligand complexes of known affinities and structures. This method employs a “protomol”, which is an idealized active site, as a target to generate presumed poses of molecules or molecular fragments. The protomol is employed as a mimic of the ideal interactions made by a perfect ligand to the active site of the protein. This molecular-similarity based alignment allows for optimization of potentially favorable molecular interactions, such as those defined by van der Waals forces and hydrogen bonds. In the present work, the protomol was defined by optimizing the threshold and bloat values to 0.5 and 0, respectively, to create a protomol that adequately described the binding pocket of interest. The extent of the protomol and its degree of coverage of an active site are controlled by these two parameters: the threshold value indicates the amount of buried-ness for the primary volume used to generate the protomol, and the bloat parameter determines the number of Ångstroms by which the search grid beyond that primary volume should be expanded. It is generally better to err on the side of a small protomol than on a protomol that is too large . All parMolecular interactions, for the docking results, are reported according to the Sampatrilat (PDB code 6F9V) amino acid sequence numbering; for comparisons between different sequence numbering in studies referred here See ). The soFor the first time, reported herein, potential interactions between the naturally produced peptides from whey and ACE have been investigated, using a molecular docking approach. Peptides, IPP, IIAE, LIVTQ, and LVYPFP formed strong H bonds and salt bridge interactions with residues in the active site of human ACE. Moreover, a comparison with commercial ACE inhibitory drugs showed that the natural peptides interacted similarly to the drugs mimicking the same interactions with ACE active site residues. This study provides strong evidence for the ACE inhibitory activity of milk derived peptides, which have not been tested in vivo before. The results of this study, of novel milk derived whey peptides, could lead to the production of novel ACE inhibitors.


Acute respiratory distress syndrome and subsequent respiratory failure remains the leading cause of death (>80%) in patients severely impacted by COVID-19. The lack of clinically effective therapies for COVID-19 calls for the consideration of novel adjunct therapeutic approaches. Though novel antiviral treatments and vaccination hold promise in control and prevention of early disease, it is noteworthy that in severe cases of COVID-19, addressing “run-away” inflammatory cascades are likely more relevant for improvement of clinical outcomes. Viral loads may decrease in severe, end-stage coronavirus cases, but a systemically damaging cytokine storm persists and mediates multiple organ injury. Remote ischemic conditioning (RIC) of the limbs has shown potential in recent years to protect the lungs and other organs against pathological conditions similar to that observed in COVID-19. We review the efficacy of RIC in protecting the lungs against acute injury and current points of consideration. The beneficial effects of RIC on lung injury along with other related cardiovascular complications are discussed, as are the limitations presented by sex and aging. This adjunct therapy is highly feasible, noninvasive, and proven to be safe in clinical conditions. If proven effective in clinical trials for acute respiratory distress syndrome and COVID-19, application in the clinical setting could be immediately implemented to improve outcomes. The global COVID-19 pandemic, announced by the World Health Organization on March 11, 2020, characterizes the clinical manifestation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection evokes pneumonia, fever, and strong inflammatory responses that can progress to acute respiratory distress syndrome (ARDS) . It has Our current understanding is that SARS-CoV-2 infection relies largely on the virus binding to membrane-bound angiotensin converting enzyme-2 (ACE2) and priming by transmembrane serene protease-2 (TMPRSS2) . Both prSurgical procedures requiring prolonged cessation of blood flow to the lungs can cause ischemia-reperfusion (IR) injury and manifest ARDS. For example, lung IR is observed in patients after pulmonary resection/lobectomy procedures, where the lung is collapsed (atelectatic) and hypoperfused for the surgical duration (∼1–2 h), followed by rapid reinflation and reperfusion of the lung tissue. In IR, tissue cell necrosis initiates the release of damage-associated molecular patterns (DAMPs), pro- and anti-inflammatory cytokines, and ROS, as well as recruitment of circulating macrophages and neutrophils that increase local vascular permeability and generate prothrombotic factors . The casThere has been large focus, mainly in surgical arenas, on developing clinical strategies to ameliorate IR- and inflammation-induced ARDS . A promiRIC is the application of brief, nonlethal bouts of an ischemic challenge before (preconditioning), during (perconditioning), or after (postconditioning) a severe stress in a distant organ . IschemiThe first report of the effectiveness of RIC in humans was against IR-induced endothelial dysfunction . A manuaThere are several animal models whereby RIC has shown promise in protecting against lung injury arising from different stressors  with similar pathological mechanisms to COVID-19 ARDS. Hindlimb ischemic preconditioning in a porcine model of pulmonary IR was shown to completely protect against impaired gas exchange and pulmonary hypertension, as well as the systemic rise in the levels of macrophages and IL-1β, but not IL-6 or ROS . In adulIn a shock model using Sprague-Dawley rats, hemorrhagic shock and resuscitation was used to induce acute lung injury, which coincided with lung inflammation, edema, leukocyte infiltration, and increases in plasma IL-6 . HindlimSimilar promise with remote conditioning has been shown in humans. Remote ischemic preconditioning of the leg improved gas exchange and lowered lung oxidative stress following lung lobectomy . LikewisThough these models of IR and inflammation-mediated lung injury are similar to the pathophysiology of COVID-19, no studies have directly assessed the efficacy of RIC on pneumonia-induced lung injury. Indirect evidence in patients with obstructive sleep apnea provides insight into the promising potential of using RIC to protect against COVID-19 ARDS. Patients with obstructive sleep apnea undergo systemic intermittent hypercapnic hypoxia from repeat apneas. Though intermittent hypoxia is different from limb ischemic conditioning, similar protective mechanisms may be manifested systemically, as both stimuli have been shown to induce similar protective cellular signaling pathways mediated by reactive oxygen species, hypoxia-inducible factor 1, protein kinase C, nitric oxide, antioxidant enzymes, and the mitochondrial K-ATP channel, via common triggers such as adenosine, adrenergic, and opioid receptor activation –53. InteThe initial reports of COVID-19 identified the condition as a respiratory illness. Although it is true that significant clinical symptoms, including respiratory distress, and the use of the respiratory system as a means of entry, support a critical role for the lungs in the pathogenesis of COVID-19, emerging and established evidence points to a more systemic disease. Arguably the most widely affected system besides the lungs is the cardiovascular system. Severely ill patients experience multiorgan endothelitis and other vascular-based disorders, consequent of the systemic propagation of the cytokine storm from lung infection, as well as from direct endothelial viral infection . FurtherAn emerging challenge in the clinical management of patients with COVID-19 is the development of widespread thrombosis. Patients with COVID-19 exhibit elevated markers of hypercoagulation that correspond with negative outcomes, and deep vein thrombosis and pulmonary embolism are reported at unusually high rates . EquallyIn addition to the blood clotting disorders that characterize SARS-CoV-2 infection, impairment to the microcirculation has been reported in patients with COVID-19 . VasculaThe prevalence of COVID-19 is generally comparable between men and women across the globe . HoweverSex-specific treatment strategies have emerged as an important consideration in the clinical management of disease. Sex differences in the response to RIC are inconsistent and not widely studied, which makes it difficult to determine if this treatment would equally benefit males and females. In a recent investigation, isolated male rat hearts (Langendorff) were treated with the plasma of young men and women before and after ischemic conditioning of the arm , which iThe seminal discovery in humans by Loukogeorgakis et al.  that RICMost experimental models have tested the benefits of ischemic conditioning under focused disease conditions, absent of comorbidities. The human condition is typically much more complex and this challenge is seen in patients with COVID-19. The most severe clinical conditions of SARS-CoV-2 infection have a higher likelihood of manifesting in older patients, often with a number of concomitant health challenges –6, 14. SThe benefits of pre-, per-, and post-conditioning have been demonstrated in a number of animal models subjected to different stressors across a multitude of organ systems. Despite clear evidence in nonhuman animals, the clinical translation of RIC has proven problematic and is a clear limitation of its potential. The RIC-STEMI randomized clinical trial of 448 patients diagnosed with ST-elevated myocardial infarction (STEMI) showed that RIC decreased cardiac mortality and hospitalization for heart failure . By contPneumonia-induced ARDS and subsequent respiratory failure remains the leading cause of death in patients severely impacted by COVID-19, requiring novel adjunct therapeutic approaches. RIC of the limbs has shown potential in recent years to protect the lungs (and other organs) against pathological conditions similar to that observed in COVID-19 B. IschemThis work was supported by a Natural Science and Engineering Research Council of Canada (NSERC) Canada Graduate Scholarship and a Canadian Institutes of Health Research (CIHR) Fellowship (to A.V.I.); NSERC Discovery Award No. 04732 and CIHR Project Grant 437970 (to W.G.P.); NSERC Discovery Grant No. 04287, Canada Foundation for Innovation, and an Ontario Early Research Award (to P.J.M.).No conflicts of interest, financial or otherwise, are declared by the authors.A.V.I. prepared figures; A.V.I., P.J.M., and W.G.P. drafted manuscript; A.V.I., P.J.M., and W.G.P. edited and revised manuscript; A.V.I., P.J.M., and W.G.P. approved final version of manuscript.


Background and Purpose: Recent findings suggested that non-stenosing atherosclerosis (NSA) may play an important pathogenic role, especially in cryptogenic strokes. Furthermore, arterial stiffness has been suggested to be a useful tool in identifying patients with embolic stroke of undetermined source (ESUS) with poor neurological prognosis. In this view, the aim of our study was to assess the association between carotid NSA and arterial stiffness in ESUS patients, in order to better define the cardiovascular risk profile of this subgroup of patients.Methods: We enrolled 100 patients with ESUS  and 48 patients with ischemic stroke from atherosclerosis. All patients underwent clinical and neuroimaging examination. A 24-h heart rate and blood pressure monitoring was performed in order to evaluate systolic, diastolic and mean blood pressure, pulse pressure, and arterial stiffness index (ASI).Results: NSA was present in 48 patients. In comparison with non-NSA-ESUS, in NSA-ESUS the mean age was higher, neurological deficit was more severe, hypertension, and diabetes were more common; systolic blood pressure, pulse pressure, and ASI were higher. In particular NSA-ESUS had ASI levels similar to strokes due to atherosclerosis.Conclusions: Our findings shed light on specific cardiovascular risk profiles underlying different subtypes of ESUS, suggesting the presence of increased arterial stiffness in NSA-ESUS patients with a risk factors profile similar to strokes due to atherosclerosis. Embolic strokes of undetermined source (ESUS) represent one-third of ischemic strokes. Although embolism is presumably the underlying mechanism of these strokes, the current definition of ESUS seems to be too broad , includiIt is well-known that arterial stiffness is associated with atherosclerotic burden and incident cardiovascular events  and may In this view, the aim of our study was to assess the presence of the common cardiovascular risk factors and especially arterial stiffness, in patients with ESUS associated with non-stenosing extracranial/intracranial carotid atherosclerotic plaques, in order to define a more complete cardiovascular risk profile in this subgroup of patients.The data that support the findings of this study are available from the corresponding author upon reasonable request. We enrolled 100 patients, admitted consecutively to our Stroke Unit  for ESUS, defined according to criteria proposed by the Cryptogenic Stroke/ESUS International Working Group (All the patients underwent carotid ultrasonography procedure by means of iE33 ultrasound system (probe 1.8–3.6 MHz) : in all patients ICA stenosis degree (ECST method)  and plaqPlaques were classified according to the five type classification : Type 1 BP monitoring was performed using validated oscillometric devices , in the first 24 h from the admission in the Hospital. BP was recorded for 24 h every 30 min, between 06:00 h to midnight, and hourly, from midnight to 06:00 h. For inclusion in the study, at least 80% of valid BP and heart rate measurements for each subject were needed.The following parameters were evaluated: SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; PP, pulse pressure; and HR, heart rate.The 24-h ABPM data were divided into daytime (06:00–22:00 h) and night-time (22:00–06:00 h) values. Dipping pattern was defined as follows: dippers had a ≥10% but <20% fall in SBP; non-dippers had an SBP fall <10% but >0%; extreme dippers had an SBP fall ≥20%; and reverse dippers had an SBP increase .By plotting the individual values of SBP and DBP measurements, obtained through 24-h non-invasive monitoring, the linear regression slope of DBP on SBP was obtained and assumed as a global measure of arterial compliance, and its complement (1 minus the slope), named ASI, has been taken as a measure of arterial stiffness . Normal post-hoc test were performed: Dunn's test for Kruskal-Wallis, multiple unpaired t-test with false discovery rate correction for ANOVA and multiple Fisher exact test with false discovery rate correction for approximation of Fisher exact test. Multivariate logistic regression was evaluated, and the stepwise procedure based on Akaike's criterion was performed. Odds Ratio (OR) and their confidence intervals (CI) were calculated. A p-value below 0.05 was considered statistically significant.Statistical analysis was performed using the GraphPad Instat computer software  and R version 3.6.2. All results were presented as mean ± SD values. Normal distribution of quantitative variables was preliminary tested using the Kolmogorov–Smirnov test, instead, the homoscedasticity with Bartlett's test. Kruskal-Wallis or ANOVA tests, according to the normality distribution and the homoscedasticity, where used to compare mean differences of NSA groups with ATS confirmatory group; for qualitative variables, an approximation of Fisher exact test was used. When these tests were statistically significant In our study, we enrolled 100 patients with ESUS. As a confirmatory group we also evaluated a group of 48 patients with stroke due to atherosclerosis, comparable with NSA-ESUS in terms of age and sex see .NSA-ESUS patients and atherosclerotic stroke patients were older than non-NSA patients. The neurological deficit was more severe in patients with atherosclerotic stroke than in non-NSA patients. In both NSA-ESUS and atherosclerotic strokes, we found a substantially greater proportion of subjects with hypertension; furthermore, the laboratory parameters showed increased levels of HbA1c in NSA-ESUS in comparison with non-NSA group and increased levels of C-reactive protein (CRP) in strokes due to atherosclerosis in comparison with non-NSA group .Considering the hemodynamic parameters, in NSA-ESUS, SBP, ASI, and PP were significantly higher than in non-NSA patients . In compOR = 2.92 for ASI > 0.70, 95% CI 1.18–7.56), diabetes , age .Multivariate logistic regression model was constructed to determine the factors associated with NSA-ESUS. The following variables were initially evaluated: age (>65 years), clinical severity of ischemic stroke (NIHSS score ≥8), presence of hypertension, presence of diabetes mellitus, C reactive protein levels, presence of arterial stiffness (ASI > 0.70). Akaike's information criterion showed that the best subset of associated variables for NSA-ESUS were the following: ASI > 0.70 (The novel findings of our study are the following: (1) ESUS patients with NSA have a specific cardiovascular profile risk, similar to patients with stroke due to atherosclerosis, (2) NSA-ESUS patients have higher ASI values in comparison with non-NSA ESUS.Indeed, our results demonstrated that in ESUS patients arterial stiffness  is significantly associated with the presence of non-stenosant carotid plaques, independent of other risk factors.Arterial stiffness is a well-known risk factor for cardiovascular events , associaThe association between this subtype of ESUS and arterial stiffness is particularly important in order to explain some possible collateral effects of anticoagulant therapy in these patients. Indeed, in NSA-ESUS, a recent study showed that aspirin was safer than rivaroxaban, determining lower rates of major bleeding . Since pOur cohort of patients presented higher NIHSS values in comparison with other previous studies , 22, eveLastly, our cohort of patients with non-NSA-ESUS had lower levels of CRP in comparison with strokes due to atherosclerosis; instead NSA-ESUS seem to have intermediate values, but not significantly different in comparison with both non-NSA ESUS and atherosclerotic strokes. It is well-known the pathogenic link among inflammation, atherosclerosis, and arterial stiffness , thus thThis study has the following limitations: the recruitment was mono-centric, the sample size is small, there are no data about the link between arterial stiffness and possible long-term complications . In this view our study represents a hypothesis-generating research, thus further studies are needed in order to confirm our findings.Our findings shed light on specific cardiovascular risk profiles underlying different subtypes of ESUS, suggesting the presence of increased arterial stiffness in NSA-ESUS patients with a risk factors profile similar to strokes due to atherosclerosis.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 University Hospital of Siena, Italy. The patients/participants provided their written informed consent to participate in this study.MA: conception and design of the work. MA, PL, CM, FG, RT, CD, and GM: substantial contributions to the acquisition of data for the work. MA, PL, CM, CD, and AC: substantial contributions to the analysis of data for the work. MA, PL, GM, and AC: substantial contributions to the interpretation of data for the work and revising the draft of the work critically for important intellectual content. MA, PL, and AC: drafting the work. MA, PL, CM, FG, RT, CD, GM, and AC: final approval of the version to be published and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 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: In this study, we investigated the ability of fluid-attenuated inversion recovery (FLAIR) data coupled with machine-leaning algorithms to differentiate normal and epileptic brains and identify the laterality of focus side in temporal lobe epilepsy (TLE) patients with visually negative MRI.Materials and Methods: The MRI data were acquired on a 3-T MR system . After pre-proceeding stage, the FLAIR signal intensities were extracted from specific regions of interest, such as the amygdala, cerebral white matter, inferior temporal gyrus, middle temporal gyrus, parahippocampal gyrus, superior temporal gyrus, and temporal pole, and fed into a classification framework followed by a support vector machine as classifier. The proposed lateralization framework was assessed in a group of MRI-negative unilateral TLE patients  and 34 healthy controls (HCs) based on a leave-one-out cross-validation strategy.Results: Using the FLAIR data, we obtained a 75% accuracy for discriminating the three groups, as well as 87.71, 83.01, and 76.19% accuracies for HC/right TLE, HC/left TLE, and left TLE/right TLE tasks, respectively.Interpretation: The experimental results show that FLAIR data can potentially be considered an informative biomarker for improving the pre-surgical diagnostic confidence in patients with MRI-negative TLE. The maiclerosis –8. Thus,clerosis . Becauseclerosis , 11, theTo overcome shortcomings, we analyzed the utility of FLAIR imaging as an alternative modality for solving the classification and lateralization problem in MRI-negative TLE patients. We hypothesized that FLAIR data coupled with machine-learning algorithms would allow us to distinguish TLE patients from healthy controls (HCs) and determining the lateralization of MRI-negative TLE individuals. To this end, we applied a machine-learning algorithm to the FLAIR signal intensity data extracted from specific regions of interest (ROIs) such as the amygdala, cerebral white matter, inferior temporal gyrus, middle temporal gyrus, parahippocampal gyrus, superior temporal gyrus, and temporal pole. We assessed the efficiency of the proposed methodology in 19 MRI-negative right TLE (RTLE) patients, 23 MRI-negative left TLE (LTLE) patients, and 34 HCs by means of a leave-one-out cross-validation (LOOCV) strategy and with a support vector machine (SVM) as classifier.F = 0.04, p = 0.96; ANOVA). In this study, a total of 76 individuals−34 HCs  and 42 MRI-negative PET-positive unilateral TLE patients [23 LTLE  and 19 RTLE ]—were recruited at the National Center of Neurology and Psychiatry (NCNP) Hospital, Tokyo, Japan, between January 2015 and November 2017. Regarding age at examination, there were no significant differences among three groups . First, for each individual, we registered the FLAIR scan to the respective high-resolution MRI scan on the basis of linear affine transformation . Then, we applied a spatially adaptive non-local means denoising filter to the FLAIR scans through the CAT12 toolbox (http://www.neuro.uni-jena.de/cat/). Afterward, we applied a special normalization to the bias-corrected FLAIR scans using the Clinical toolbox , followed by a default FLAIR template generated from 181 individuals  . The sequences and their parameters were as follows: three-dimensional (3D) sagittal T1-weighted magnetization prepared rapid acquisition with gradient echo (MPRAGE) images ; sagittal 3D fluid-attenuated inversion recovery (FLAIR) images . Additionally, for visual assessment of potential epileptogenic lesions, both T1 and FLAIR images were reconstructed into coronal and axial slices and high-resolution coronal T2-weighted images were also obtained. The FLAIR scans were analyzed by Statistical Parametric Mapping toolbox version 12  as well as a multiclass classification model . The performance of each classification model was computed using a LOOCV strategy such that, each time, one sample was used for the test set and the remainder for training. A SVM implemented in MATLAB  was used a classifier. Using a linear SVM kernel, we are able to visualize the contribution of individual feature  to the overall classification models. As for binary classification models, the prediction performance was assessed by means of accuracy (ACC), sensitivity (SEN), specificity (SPE), and area under the receiver operating characteristic curve (ROC-AUC) metrics, whereas the classification performance for the multiclass classification model was reported in terms of accuracy and confusion matrix. In addition, to determine the contribution of each feature  in our classification models, we aggregated the SVM coefficients achieved from training sets over the LOOCV strategy and then averaged them.To validate the performance of FLAIR data for the diagnosis and lateralization of MRI-negative t-test = 0.94, p = 0.34; student t-test) and duration of disease . As for HC group, participants had no history of neurological or psychiatric diseases, no use of medication affecting the central nervous system, and no structural abnormalities on MRI. TLE was diagnosed based on the presence of focal seizures consistent with TLE and focal epileptiform discharge predominantly in temporal areas on a conventional scalp electroencephalogram (EEG) using standard international 10–20 system. All patients underwent 3-T MRI and interictal FDG PET, which were visually evaluated by experienced neuroradiologists. All TLE patients showed unilateral glucose hypometabolism including the temporal lobe in interictal FDG PET consistent with the clinical symptoms and EEG abnormalities, without any evidence of morphological abnormality on visual assessment of MRI scans. To investigate a distinct group of MRI-negative PET-positive unilateral TLE, we recruited only patients with concordant ipsilateral hypometabolism in FDG PET, and all TLE patients were drug-resistant. In TLE patients' group, there were no significant differences between RTLE and LTLE in terms of onset age .For the sake of illustration, Regarding the binary classification models, HCs were distinguished from RTLE patients with 87.71% accuracy  and from LTLE patients with 83.01% accuracy  while LTLE patients were differentiated from RTLE patients with 76.19% accuracy . To assess the impact of hippocampus on the classification performances, we recomputed the prediction accuracies after adding the hippocampus data in our prediction models. The classification results were as follow: HC/RTLE = 87.71%, HC/LTLE = 79.24%, RTLE/LTLE = 71.42%, and HC/RTLE/LTLE = 69.73%. By comparing these results with prediction results presented in section “Experimental Results”, it can be seen that including the hippocampus data in prediction models could not improve the prediction accuracies for distinguishing TLE patients from HCs and detecting the lateralization of MRI-negative TLE individuals.In this study, we selected the specific ROIs in accordance with the pathology-based knowledge which may reflect potential epileptogenic lesions, astrogliosis, and related changes . To show whether the selected regions were appropriate for FLAIR-wise classification and lateralization models, we validated the classification models using the whole-brain FLAIR signal intensities . In this case , the performance of our classification models in terms of accuracy were as follows: HC/RTLE = 82.45%, HC/LTLE = 75.47%, RTLE/LTLE = 54.76%, and HC/RTLE/LTLE = 56.57%. Furthermore, we applied principal component analysis (PCA) data reduction technique on whole-brain FLAIR data. As for PCA data reduction followed by 95% amount of total variance, we achieved the following prediction results: HC/RTLE = 75.43%, HC/LTLE = 73.58%, RTLE/LTLE = 42.58%, and HC/RTLE/LTLE = 50%. When we compared the prediction results using whole-brain FLAIR signal intensities, PCA data reduction method, and FLAIR signal intensities extracted from specific regions , it was evident that our pre-defined regions-based strategy might provide more informative data for the classification and lateralization of MRI-negative TLE individuals rather than whole-brain FLAIR data and PCA data reduction.A series of neuroimaging studies have designed and developed new techniques for the classification and lateralization of TLEs based on different brain imaging modalities. For instance, the researchers in  suggesteLateralizing the focus side of TLE is a highly relevant in drug-resistant patients. According to the previous studies , 8, MRI-Accordingly, we were motivated to assess the utility of FLAIR data, as a low-cost and widely available modality, coupled with machine-learning algorithms for addressing the classification and lateralization problem in MRI-negative TLE patients. To this end, we extracted the FLAIR signals from specific parts of brain  and then fed the data into the classification models. Using this strategy, our experimental results determined that FLAIR data can distinguish the three groups  with 75% accuracy, suggesting that FLAIR data has the potential to be considered a lower-cost alternative for the classification and lateralization of MRI-negative TLE patients.Based on the information obtained in the machine-learning stage, we specified the contribution of each region for the classification models see . This viIn the field of epilepsy neuroimaging, some studies have documented an important link between the hippocampus and epilepsy, particularly in TLE patients with hippocampal sclerosis . HoweverTo our knowledge, this is the first study to use pattern analysis of FLAIR data for the classification and lateralization of MRI-negative TLE patients. Future research into the use of FLAIR markers for the diagnosis and lateralization of MRI-negative PET-positive TLE individuals is nonetheless required. Finally, the lack of detailed location of definite epileptogenic zone and surgical outcome is an important limitation of this study. On the other hand, given the evidence on the good seizure outcome after anterior temporal lobectomy in MRI-negative PET-positive TLE , 8, it iThe aim of this study was to examine the utility of FLAIR data for the classification and lateralization of TLE patients with visually normal brain MRIs. Our experimental results indicated that the FLAIR modality has remarkable functionality and could be considered an accessible, safe, low-cost, and informative predictor for the classification and lateralization of MRI-negative TLE individuals in the clinical setting.beheshtiiman@gmail.com.The data analyzed in this study is subject to the following licenses/restrictions: It is a private dataset. Requests to access these datasets should be directed to Iman Beheshti, The studies involving human participants were reviewed and approved by all participants provided written informed consent, and the study was approved by the Institutional Review Board at the National Center of Neurology and Psychiatry Hospital. The patients/participants provided their written informed consent to participate in this study.IB and DS designed research and performed research and wrote the paper. NM, YK, YS, and NS collected the data and performed PET interpretation. HM supervised the study. 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.


Plasmodium is a frequent global threat in malaria eradication programmes, highlighting the need for new anti-malarial drugs and efficient detection of treatment failure. Plasmodium falciparum culture is essential in drug discovery and resistance surveillance. Microscopy of Giemsa-stained erythrocytes is common for determining anti-malarial effects on the intraerythrocytic development of cultured Plasmodium parasites. Giemsa-based microscopy use is conventional but laborious, and its accuracy depends largely on examiner skill. Given the availability of nucleic acid-binding fluorescent dyes and advances in flow cytometry, the use of various fluorochromes has been frequently attempted for the enumeration of parasitaemia and discrimination of P. falciparum growth in drug susceptibility assays. However, fluorochromes do not meet the requirements of being fast, simple, reliable and sensitive. Thus, this review revisits the utility of fluorochromes, notes previously reported hindrances, and highlights the challenges and opportunities for using fluorochromes in flow cytometer-based drug susceptibility tests. It aims to improve drug discovery and support a resistance surveillance system, an essential feature in combatting malaria.Drug-resistant  Plasmodium falciparum is highly virulent [Plasmodium species. Given the occurrence of anti-malarial drug resistance [P. falciparum, either laboratory strains or field isolates, and microscopic examination to assess the growth of the cultured Plasmodium parasites [Plasmodium parasites are tedious and time-consuming. Moreover, interrater variability among the microscopists largely affects the interpretation, emphasizing a need for rapider and simpler assays that retain accuracy.Malaria caused by the parasitic protozoan virulent  and a lesistance , 2, a suarasites –6. DespiPlasmodium, lack nuclear DNA, detection of malarial DNA is thus able to distinguish parasitized erythrocytes from non-infected erythrocytes, especially those in a leukocyte-free culture of Plasmodium parasites. At present, many DNA- and RNA-binding fluorochromes are commercially available, and they are divided into two types: cell membrane permeable and non-permeable  and a measurement of parasite growth. Since then, Giemsa-based microscopy has become the gold standard for the diagnosis and clinical management of malaria [The use of a light microscope paved the way to discovering many pathogenic microorganisms causing human diseases, advancing our knowledge of medicine. Without staining with a dye, Alphonse Laveran first noticed an unknown microorganism with actively mobile filaments in blood taken from an infected soldier under a microscope, later confirming it as a flagella . Once th malaria  and an ePlasmodium spp. have been widely used as a malaria model in the preclinical phase of anti-malarial drug development and as a tool for drug resistance surveillance. To model malaria in vitro, laboratory-adapted P. falciparum strains or field-isolated strains are cultured with human erythrocytes, mostly those of blood group O, in RPMI 1640 medium supplemented with HEPES, sodium bicarbonate, and heat-inactivated human AB serum. The in vitro or ex vivo cultured Plasmodium parasites are then incubated with serial dilutions of drug and subjected to enumerating parasitized erythrocytes (schizonts) using a microscope. Giemsa-based light microscopy is the main method used to measure the intraerythrocytic development of Plasmodium parasites [Plasmodium growth inhibition assays, a synchronous culture is treated with a drug [Plasmodium parasites is examined. There are several methods for the assessment of intraerythrocytic Plasmodium growth: morphological observation under a microscope and biochemical measurements. For morphology-based assays, the microtechnique developed by Rieckmann et al. [Plasmodium uptake of radiolabelled hypoxanthine, a nucleic acid precursor, to assess the growth inhibitory effect of anti-malarial drugs [Plasmodium growth inhibition in experiments [Plasmodium parasites in intraerythrocytic stages also actively synthesize cell membranes composed of phospholipids; thus, an assay based on incorporation of the radioisotope-labeled phospholipid precursor ethanolamine was developed [Plasmodium-specific lactate dehydrogenase [To assess the effect of a drug on the intraerythrocytic growth of malaria parasites, cultures of arasites , 10, andarasites . In Plash a drug . Then, tn et al.  is simpln et al. –18. For al drugs  or immunal drugs . Given aeriments  and in aeriments  and for eriments . In addieveloped , allowineveloped . Apart frogenase –28 and hrogenase , 30 are rogenase , 32.P. berghei-infected mouse erythrocytes. In early 1990, a fluorescence-based flow cytometer was introduced for drug susceptibility testing and parasite detection in human blood [Plasmodium-infected cells and leukocytes. Approximately 16–20 h of drug testing was performed in a 96-well plate, while fluorescence-based flow cytometry consisting of cell staining, washing and acquisition was performed within 2 h [In 1979, Howard et al.  were thean blood , 35. Comthin 2 h . Despitethin 2 h –38. ThusPlasmodium development in culture  is first prepared and subsequently treated with various doses of drug. Mostly, synchronized cultures of ring-like Plasmodium parasites are incubated with drugs, and their growth is assessed mostly at the schizont stage. Hence, prior drug treatment and heterogeneity in Plasmodium parasites may confound the interpretation. Owing to the high resolution of the emitted fluorescence intensity, flow cytometry offers an option to determine the extent to which the culture is synchronous  and 650 (red fluorescence) nm when the dye is bound to DNA or RNA, respectively, allowing discrimination between DNA and RNA. Despite cell permeability, acridine orange failed to detect  setting , suggest setting  deployedcroscope . To datetivation . In rodeod cells . Neverth berghei . HoweverPlasmodium growth in vitro [Coriphosphine O is a dsDNA intercalating and RNA loop-binding fluorochrome. In addition to its excitation by common 488-nm argon ion lasers, an advantage in using Coriphosphine O is its ability to emit light with two distinct spectra: green and orange fluorescence is emitted when it is bound to DNA and RNA, respectively. Moreover, given the large difference in the positions of the band maxima of the absorption and emission spectra (known as the Stokes shift) after it binds to nucleic acids, coriphosphine O is hence able to distinguish parasitized erythrocytes, especially those in the early ring-form stage, from non-infected cells at high resolution . In a stin vitro . The resP. falciparum-infected erythrocytes in all four major stages and that its fluorescence intensity depended on the intraerythrocytic stage of P. falciparum development. Of note, the VSG-based flow cytometric assay failed to distinguish gametocytes from schizonts [P. falciparum development in erythrocytes and the growth inhibitory effect of anti-malarial drugs in vitro. Compared to the use of other cell-permeant fluorochromes, the use of VSG is a relatively simple and fixation-free method that enumerates malaria-infected erythrocytes and assesses intraerythrocytic development in culture and in anti-malarial drug susceptibility assays. However, given DNA- and RNA-binding ability, the interpretation of VSG + cells is likely confounded by the presence of reticulocytes, nucleated erythrocytes and leukocytes. Moreover, VSG was not able to distinguish the early gametocyte stage in cultures containing schizonts. Thus, further validation and comparison with other fluorochromes are required to conclude its superiority.ViSafe Green (VSG) is an environmentally safe nucleic acid-binding fluorescent dye that has been applied for visualizing DNA and RNA in agarose and polyacrylamide gels. VSG can be activated by 250–300 nm (UV) light, and its emission exhibits a spectrum similar to that of ethidium bromide . VSG is,chizonts . The obtSome nucleic acid-binding fluorochromes require an additional step of cell membrane permeabilization. Aldehyde and ethanol are commonly used for fixation and permeabilization. Since both chemicals inevitably alter cell structure, morphological observation of malaria parasites is impossible after cell membrane permeabilization, a drawback of cell-impermeant fluorochromes.Plasmodium nucleic acids could be stained with PI after fixation with paraformaldehyde and glutaraldehyde [in vitro drug susceptibility tests of P. falciparum, the minimum concentration of drugs that inhibit Plasmodium parasite growth could be calculated based on a decrease in fluorescence intensity, and the results correlated with microscopy observations [P. falciparum trophozoites from clinical blood samples for whole genome studies without host genome contamination [Propidium iodide (PI) is an intercalating dye that binds to both DNA and RNA without nucleotide preference. Although unbound PI in solution can be excited, the emitted fluorescence signal is enhanced 20- to 30-fold after intercalation between nucleic acids. When bound to DNA, the excitation/emission maxima of PI are 535 nm (green)/617 nm (orange–red). PI has been widely applied for many purposes. First, since PI is unable to permeate the cell membrane, it can differentiate viable cells from nonviable cells that have lost membrane integrity. Second, the fluorescence intensity of PI is proportional to the DNA content, allowing cell cycle analysis. However, PI also binds to RNA; thus, it is necessary to eliminate RNA using RNase prior to staining with PI. Regarding applications of PI for malaria research, aldehyde . In in vrvations . Moreovemination .max = 497 nm) and emitting green light (λmax = 520 nm). SGI reportedly binds to single-stranded DNA and RNA, then emitting a fluorescent signal approximately 10-fold less intense than that for dsDNA binding [SYBR Green I (SGI) is an asymmetrical cyanine dye (synthetic dyes belonging to the polymethine group) that was originally used for visualizing nucleic acids in gel electrophoresis, quantifying DNA in solution and detecting amplified products in quantitative real-time PCR. SGI interacts noncovalently with dsDNA via intercalation or minor groove binding . Upon DN binding , 66.Plasmodium parasites, there are two ways to use SGI-based flow cytometric analysis: with or without cell fixation. Izumiyama reported that without cell fixation, SGI staining of P. falciparum-infected erythrocytes was able to differentiate intraerythrocytic stages based on the fluorescence intensity at the wavelength of maximum intensity. Ring-form and young trophozoites exhibited the lowest fluorescence intensity, whereas mature schizonts had the highest fluorescence intensity. The developmental stages exhibiting an intermediate level of fluorescence intensity were the late trophozoite and young schizont stages [P. falciparum strains, fixation-free, SG-based flow cytometry was applicable to first round invasion-derived parasitaemia of clinical isolates [P. falciparum merozoite counting assay allowed the assessment of chloroquine effects on schizogony [P. falciparum [Plasmodium stages, limiting the assessment of parasite growth [Regarding cell analysis, SGI was utilized for enumeration of viable cultured bacteria  and detet stages . The fixt stages . In addiisolates . For thehizogony  and reinhizogony , 69. Morlciparum . Howevere growth . TherefoP. falciparum. Similar to other cell-impermeant dyes, fixation with formaldehyde is inevitable. Moreover, subsequent permeabilization with Triton X-100 (TX100) was deployed, ending 24 h before YOYO-1 staining [Plasmodium development [in vitro culture of Plasmodium parasites but is not yet applicable for field isolates, largely due to its RNA-binding activity in reticulocytes.YOYO-1, a cyanine dye, is a homodimer of oxazole yellow (known as YO) and binds to DNA via intercalation . In solustaining . The fluelopment . When thelopment . In bothPicoGreen, an intercalating, cell-impermeant fluorochrome, specifically binds to dsDNA but not to ssDNA, RNA, or free nucleotides. The PicoGreen-dsDNA complex can be excited by 480-nm light and maximally emits at 520 nm. In contrast to its high fluorescence when bound to DNA, unbound PicoGreen emits fluorescence with a very low intensity. PicoGreen is resistant to photobleaching, which is an irreversible fading of fluorescence caused by light exposure; therefore, intra-assay variance is avoided owing to the high stability of the dye. Thus, it is suitable for high-throughput, multiwell-formatted assays and microplate readers. The use of PicoGreen in anti-malarial chloroquine assays gave a similar growth inhibitory concentration as the standard radioactive hypoxanthine uptake assay , 76. GivPlasmodium inside the host cells. In most living eukaryotic cells, functioning mitochondria maintain their membrane polarization, whereas in metabolically inactive cells, nonviable cells lose such polarization [Plasmodium parasites could be detected based on mitochondrial function. Fluorescent dyes that bind to the polarized mitochondrial membrane, herein called mitochondrial dyes, are able to differentiate between living and dead cells [Plasmodium parasites, incubation at 37 °C is inevitable. Since nonviable cells have a compromised cell membrane, cell-impermeant fluorochromes are able to cross the compromised cell membrane and intracellularly bind to nucleic acids. Based on these properties, an approach to overcome this complicated protocol is to combine cell-permeant and cell-impermeant fluorochromes, especially those maximally emitting at different wavelengths. For instance, Hoechst 33,342, SYBR Green I or ViSafe Green can be combined with propidium iodide. Moreover, SYTOX® blue, a high-affinity nucleic acid dye that fluoresces, is able to enter only cells with compromised cell membranes. Thus, it has the potential to detect dead cells. To the best of my knowledge, there has been no report in which these combinations of fluorochromes have been deployed for Plasmodium-infected erythrocytes.A major drawback of the aforementioned DNA/RNA-staining fluorochromes is the inability to distinguish live and dead parasites owing to remnant DNA and/or RNA of rization . Thus, lad cells . MitoTraad cells . AlthougThe emergence of anti-malarial drug resistance highlights the importance of drug susceptibility tests by which the existence and spread of resistant parasites can be detected and tracked. At present, given the availability of a compact, transportable flow cytometer, a combination of SYBR Green I and dihydroethidium, both cell permeant, has been applied for the detection of drug resistance in malaria endemic areas in Papua Province, Indonesia  and ThaiPlasmodium-infected erythrocytes can be directly stained with a cell-permeant fluorochrome. Neither cell fixation nor harvesting are required. Given fluorescence enhancement (increasing fluorescence intensity or a Stokes shift upon binding to nucleic acids), the stained cells can be directly exposed to laser light without the removal of unbound fluorochrome. Thus, a washing step is not needed. Highly selective DNA binding of the fluorochrome will minimize the effects of confounding factors derived from RNA binding in reticulocytes. Since several wells are analysed, the fluorochrome must resist photobleaching. In addition, the high resolution of fluorescence intensity allows further discrimination of ring-form trophozoites, trophozoites and schizonts, characterizing stage-specific drug resistance.Various doses of anti-malarial drugs need to be tested; thus, the assay required a protocol suitable for a multiwell operation. Here, key characteristics of fluorochromes are proposed for their use in flow cytometer-based drug susceptibility assays Fig. . To alloPlasmodium-infected erythrocytes [Plasmodium. As discussed in viable parasite detection, there are two types of fluorescent dyes: one binds to the polarized mitochondrial membrane, and the other is a cell-impermeant dye capable of crossing a compromised cell membrane. Combinations of mitochondrial or cell-impermeant dyes with cell-permeant dyes will allow assessment of cidal effects in addition to growth inhibition.However, there are two major hindrances in anti-malarial drug sensitivity tests. First, the use of nucleic acid-binding fluorochromes will lose accuracy owing to fluorescence signals of RNA in reticulocytes and remnant DNA (Howell-Jolly bodies) in some erythrocytes, both in erythrocytes or leukocytes in the peripheral blood of human individuals. To exclude such artifacts in blood, deploying fluorochrome binding specifically to RNA, antibody specific to transferrin receptor, highly expressed on immature enucleated erythrocytes and nucleated erythroid cells, or CD45, a common leukocyte antigen, allows more accurate identification of hrocytes , 58. SecP. berghei gametocytes with DRAQ5 was accomplished at a higher temperature [DRAQ5™ is a cell-permeable, DNA-binding anthraquinone. Given the lipophilicity of DRAQ5, it can cross phospholipid-enriched membranes without cell fixation . Althougperature . Since Dperature . MoreovePlasmodium-infected erythrocytes [Formaldehyde and glutaraldehyde partially solubilize lipids on the cell membrane , consequhrocytes  and basohrocytes . TX100 ihrocytes . Followihrocytes . Recentlhrocytes . OverallPlasmodium-infected erythrocytes. Owing to the larger amount of nuclear DNA in leukocytes, a possible solution is to search for fluorochromes exhibiting a wider range of fluorescence intensities (higher resolution); leukocytes and Plasmodium-infected erythrocytes emit fluorescence with highly different intensities. However, the invention of fluorochrome derivatives that are cell permeable, have high fluorescence enhancement, are selective for DNA binding and have fluorescence that quenches just slightly would further increase the use of flow cytometry in malaria research.Despite many successful applications of fluorochrome-based flow cytometric assays in malaria research, none of them has become a widely-used method. High-cost and multistep processes, in contrast to the need for affordable and user-friendly tools, likely account for the restricted use of these assays in the malaria field. Recently, in attempts to develop low-cost, easy-to-use flow cytometers, a portable, compact flow cytometer and a microfluidic chip equipped with fluorescence readers have been invented , 90. ThuPlasmodium-infected erythrocytes and discrimination of P. falciparum growth in drug susceptibility assays. Studies deployed fluorochromes, which are capable and incapable of crossing the cell membrane to bind nucleic acids, for assessment of anti-malarial drug efficacy. Depending on purpose of study, the following guideline is proposed; (1) a combination of dihydroethidium with Hoechst 33,342 or thiazole orange is for drug susceptibility test, and discrimination of parasite maturation stage and viability, (2) a combination of SYTO61 with dichlorofluorescin is for assessment of oxidative stress, (3) a combination of Coriphosphine with MitoTracker is for discrimination of viable and nonviable Plasmodium parasites, and (4) a combination of dihydroethidium with thiazole orange is for study of transcriptiome. However, the previously reported hindrances, including the complexity of the process, inability to distinguish live and dead parasites, and low resolution and low quenching of fluorochromes, limit their widespread use in the aforementioned applications. To overcome these hindrances, the review highlights the key features to select and opportunities to use fluorochromes in flow cytometer-based drug susceptibility tests.The various uses of nucleic acid-binding fluorochromes and flow cytometry include enumeration of


Social cognition includes mental operations essential for functional social interactions, and several studies revealed an impairment of social cognition abilities in patients with Multiple Sclerosis (MS). These deficits have been related to global and focal gray matter atrophy as well as microstructural white matter damage. Although some studies reveal a correlation between social cognition and task-based functional magnetic resonance imaging (MRI), no studies to date have explored the association between brain resting-state functional connectivity (RS-FC) abnormalities and several measures of social cognition in MS. The aim of this explorative study was to assess the contribution of RS-FC abnormalities of major brain networks to social cognition in MS patients. Clinical, neuropsychological, and MRI data were collected from 41 non-depressed and cognitively preserved relapsing-remitting MS patients  and 25 matched healthy controls (HCs). The ToM Pictures Sequencing Task (TMPS) and the Reading the Mind in the Eyes Task were employed to evaluate social cognition. All participants underwent a structural MRI and RS functional MRI 3T protocol. Regional gray matter atrophy was measured, and FCs of the default mode (DMN), right and left fronto-parietal, executive (EN), salience, cerebellar, and limbic (LN) networks were evaluated by independent component analysis (ICA). Differences on TMPS were found between MS patients and HC (MS < HC). In the MS group, associations were found between right middle temporal gyrus FC (in the DMN) and reciprocity subscale of TMPS, posterior cingulate cortex (PCC) FC (in the DMN) and first-order false-belief subscale of TMPS, cingulate gyrus FC (in the EN) and TMPS as well as reciprocity subscale of TMPS, and right superior temporal gyrus (in the LN) and reciprocity subscale of TMPS. All detected RS-FC changes did not co-localize with regional gray matter atrophy. The results suggest an association between social cognition and RS-FC changes of DMN, EN, and LN in MS. Future studies should further explore the possible adaptive or maladaptive mechanisms of these FC abnormalities in MS. Social cognition includes mental operations essential for functional social interactions  studies revealed a core network, including the medial prefrontal cortex and bilateral temporo-parietal junction, which are consistently activated independently from the type of the instrument employed to assess social cognition  fMRI studies have explored cognitive and affective ToM in MS. RS fMRI represents a unique method to investigate brain networks with minimal bias, and it has been employed in several studies in MS ; 25 Healthy controls (HCs) were recruited from a large HC database created in our Institution. As for the inclusion criteria, patients had to have a diagnosis of MS according to the revised McDonald criteria  scanner equipped with an eight-channel parallel head coil. The following images were acquired:3); (3) RS-fMRI consisting of 240 volumes of a repeated gradient-echo echo planar imaging T2*-weighted sequence . During the functional scan, subjects were asked to stay motionless, awake, and relaxed, and to keep their eyes closed.(1) Proton density (PD)/T2 weighted ; (2) high-resolution 3D-T1  blinded to the patients’ clinical characteristics. The Medical Image Processing, Analysis, and Visualization (MIPAV) software , EN, SLN, LN, and CN components. The resulting t-maps were thresholded at p = 0.05 (Bonferroni corrected over the entire brain). From these, an inclusive mask was also created from the HC group maps and used to define a new search volume for within-network, two-group comparisons. The voxel-wise comparisons between the two groups were indeed performed with two-sample t-test over the search volume. All the comparisons were made with gender and age included as covariate of no interest. To correct the resulting t-maps for multiple comparisons, regional effects within the search volume were considered significant only for compact clusters after the joint application of a voxel- and cluster-level threshold. The cluster-level threshold was estimated non-parametrically with a randomization approach: starting from an initial (uncorrected) threshold of p = 0.001 applied to all voxels, a minimum cluster size was calculated that protected against false-positive clusters at 5% after 1,000 Monte Carlo simulations . ICA z-scores express the relative modulation of a given voxel by a specific ICA component and hence reflect the amplitude of the correlated fluctuations within the corresponding functional connectivity network.Standard image data preparation and preprocessing, statistical analysis, and visualization were performed with the software BrainVoyager QX . Data preprocessing included the correction for slice scan timing acquisition, a three-dimensional rigid-body motion correction based on a six-parameter rigid body alignment to correct for minor head movements, and the application of a temporal high-pass filter with cut off set to three cycles per time course. Translational motion parameters were verified to be always less than one functional voxel for all included participants. Structural and functional data were coregistered and spatially normalized to the Talairach standard space using a 12-parameter affine transformation. During this procedure, the functional images were resampled to an isometric 3-mm grid covering the entire Talairach box. Single-subject and group-level independent component analysis (ICA) were carried out on the pre-processed functional time series using two plug-in extensions of BrainVoyager QX, implementing fast ICA algorithm and the self-organizing group-level ICA algorithm, respectively. For each subject, 40 independent components, corresponding to one sixth of the number of time points, were extracted. All single-subject component maps from all subjects were then “clustered” at group level, resulting in 40 single-group average maps that were visually inspected for recognition of the main physiological RS components. The sign-adjusted ICA components of all subjects were then submitted to a second-level, multi-subject random-effects two-way ANOVA that treated the individual subject map values as random observations at each voxel  was performed (SPSS Statistics version 25.0).A Kolmogorov–Smirnov test was used to verify normal distribution of demographic, clinical, and conventional MRI variables. Between-group comparisons were performed using the Mann–Whitney and chi-square tests, as appropriate. Correlations between scores on social cognition tasks and conventional MRI variables were assessed using Spearman’s rank correlation coefficient. A p < 0.001); see Clinical, demographical, and MRI characteristics of HC and MS patients groups are described in Comparison of RS-FC between MS patients and HC: (1) DMN: MS patients showed a decreased RS-FC in the PCC and an increased RS-FC in the right middle temporal gyrus; (2) RFPN: MS patients showed an increased RS-FC in the right middle temporal gyrus; (3) LFPN: MS patients showed a decreased RS-FC in the left middle temporal gyrus; (4) EN: MS patients showed a decreased RS-FC in the right precentral gyrus and in the cingulate gyrus; (5) SLN: MS patients showed a decreased RS-FC in the right middle temporal gyrus; (6) LN: MS patients showed an increased RS-FC in the right and the left superior temporal gyrus; and (7) CN: no significant differences were found between HC and MS patients .p = 0.0480); negative association between RS-FC in the PCC and score on first-order false-belief ToM subscale of TMPS ; (2) EN (see p = 0.0441); negative association between RS-FC in the cingulate gyrus and score on reciprocity subscale of TMPS ; (3) LN , while first-order ToM subscale of TMPS was positively correlated with FC in the right precentral gyrus in the EN .Correlations between RS-FC networks abnormalities and social cognition tasks in MS patients: (1) DMN see : positiv) EN see : negativ) LN see : negativAll detected RS-FC abnormalities did not co-localize with regional GM atrophy.x = 11, y = −30, z = −5, K = 573; left thalamus: x = −8, y = −26, z = −5, K = 1,128), and the superior frontal gyrus . No areas of regional GM volume reduction were found in HC when compared to MS patients. No correlations between social cognition tasks and regional GM volume were found in MS patients.MS patients, when compared to HC, showed significant regional GM loss in the following areas: the thalamus, bilaterally  and the low sample size of previous studies might explain these discrepancies  may overlap. Therefore, future studies could further expand our results by employing questionnaires and tools that measure multiple domains of social cognition and that are able to better discriminate between these processes.p-values were considered. Moreover, while we employed two tests that evaluated social cognition in non-verbal modality, we could not assess the relationship between the RS-FC of cognitive-related brain networks and social cognition tasks in verbal modality. This issue should be investigated in future studies. Finally, our sample included MS patients with a medium disease duration, with no cognitive impairment, and with a relatively low lesion load; because of the nature of our cross-sectional study, we could not evaluate the evolution of the associations with time, in later stages of the disease. Future research should be conducted by taking into account the limitations and the methodological weakness of the present study; we believe that the use of a larger sample, employing statistical corrective measures to rule out the possible presence of a type 1 error, and using multiple psychological tests that are able to assess several components of social cognition and differentiate between the several processes that are part of social cognition might confirm and expand our novel findings.This study is not exempt from limitations. First, we were not able to recruit an equal number of patients and controls . Moreover, when testing group differences between MS patients and HC on social cognition tasks, only total score on TMPS and score on the reciprocity subscale of TMPS remained significant after the Bonferroni correction. Nevertheless, we decided anyway to test the correlations of all social cognition tasks with RSN abnormalities because of the explorative nature of our study. In addition, since a too strict Bonferroni correction for multiple comparisons would have negatively influenced the explorative nature of the study, unadjusted In conclusion, the present study reinforces and expands the notion that patients with MS exhibit an impairment of social cognition abilities. Moreover, the performance on social cognition tasks  appears to be related to specific abnormalities in RSNs related to cognition, such as the DMN, the EN, and the LN. It must be emphasized, however, that the findings of our explorative study have to be treated with caution due to some methodological weaknesses . Future investigations should confirm our results by employing a larger number of patients, while also adopting a longitudinal design.Among the subscales of TMPS, the reciprocity score was significantly associated to three nodes in three different brain networks . The social norm of reciprocity plays a key role into maintaining cooperation among members of society; it is now considered a common feature of social cognition, since recent literature confirmed that social cognition is not only a process linked to “pure observation” of social situations, but it also includes elements of social interaction Bratman, . This coThe datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Comitato Etico Azienda Ospedaliera Universitaria Luigi Vanvitelli—AORN Ospedale dei Colli. The patients/participants provided their written informed consent to participate in this study.AG, GS, and AB conceived and designed the study. MA, SP, RC, and Ad’A collected clinical/neuropsychological data. MC collected MRI data. MA, FD, GC, and RD performed statistical and MRI analysis. AB and MA wrote the manuscript. AG, GS, and GT supervised the project. FE, SB, and FT contributed to the interpretation of the results and to the final version of the manuscript . All authors discussed the results and contributed to the final manuscript.AB has received speaker honoraria and/or compensation for consulting service from Biogen, Merck, and Genzyme. MA, GS, FD, RD, GC, RC, SP, Ad’A, MC, FE, and FT have no disclosures. SB has received speaker honoraria and advisory board fees from Novartis, Roche, Merck Serono, Biogen, Teva, Genzyme, and Almirall. GT has received compensation for consulting services and/or speaking activities from Biogen, Novartis, Merck, Genzyme, Roche, and Teva, and receives research support from Biogen Idec, Merck Serono, and Fondazione Italiana Sclerosi Multipla. AG received honoraria for speaking and travel grants from Biogen, Merck, Genzyme, Teva, Mylan, Roche, and Novartis.


Currently, the most approaches to analyze statistical associations between regions and imaging modalities rely on Pearson correlation or linear regression models. However, these models are prone to spurious correlations arising from uninformative shared variance and multicollinearity. Notably, there are no appropriate multivariate statistical models available that can easily integrate dozens of multicollinear variables derived from such data, being able to utilize the additional information provided from the combination of data sources. Gaussian graphical models (GGMs) can estimate the conditional dependency from given data, which is conceptually expected to closely reflect the underlying causal relationships between various variables. Hence, we applied GGMs to assess multimodal regional brain alterations in AD. We obtained data from N = 972 subjects from the Alzheimer's Disease Neuroimaging Initiative. The mean amyloid load (AV45-PET), glucose metabolism (FDG-PET), and gray matter volume (MRI) were calculated for each of the 108 cortical and subcortical brain regions. GGMs were estimated using a Bayesian framework for the combined multimodal data and the resulted conditional dependency networks were compared to classical covariance networks based on Pearson correlation. Additionally, graph-theoretical network statistics were calculated to determine network alterations associated with disease status. The resulting conditional dependency matrices were much sparser (≈10% density) than Pearson correlation matrices (≈50% density). Within imaging modalities, conditional dependency networks yielded clusters connecting anatomically adjacent regions. For the associations between different modalities, only few region-specific connections were detected. Network measures such as small-world coefficient were significantly altered across diagnostic groups, with a biphasic u-shape trajectory, i.e., increased small-world coefficient in early mild cognitive impairment (MCI), similar values in late MCI, and decreased values in AD dementia patients compared to cognitively normal controls. In conclusion, GGMs removed commonly shared variance among multimodal measures of regional brain alterations in MCI and AD, and yielded sparser matrices compared to correlation networks based on the Pearson coefficient. Therefore, GGMs may be used as alternative to thresholding-approaches typically applied to correlation networks to obtain the most informative relations between variables.Alzheimer's disease (AD) is characterized by a sequence of pathological changes, which are commonly assessed  Several studies suggest that information obtained from combining different imaging modalities could provide reliable markers of cerebral reserve capacity and might be used to predict and monitor the evolution of AD and its relative impact on cognitive domains in pre-clinical, prodromal, and dementia stages of AD [see e.g., reviews  is characterized by a range of pathological brain alterations that can be assessed Up to date, multimodal studies employ one of the following approaches:Correlation of pathology maps on a voxel level  to compare differences in other imaging modalities , which are able to estimate the T1-weighted MRI). Based on our previous results with only six representative brain regions , glucose metabolism (FDG-PET), and gray matter volume (http://adni.loni.usc.edu). The ADNI was launched in 2003 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies, and non-profit organizations, with the primary goal of testing whether neuroimaging, neuropsychological, and other biological measurements can be used as reliable in vivo markers of Alzheimer's disease pathogenesis. A complete description of ADNI and up-to-date information is available at http://www.adni-info.org. For this study, 529 subjects with amnestic mild cognitive impairment (MCI), 189 patients with Alzheimer's dementia (AD), and 254 cognitively healthy control subjects (CN) were selected from the ADNI-GO, ADNI-2, and ADNI-3 extensions of the ADNI project, based on the availability of concurrent structural MRI, FDG-PET, amyloid-sensitive AV45-PET, and neuropsychological assessments. In ADNI, two MCI subgroups exist, which only differ by the less severe impairment of memory function for early MCI (EMCI) compared to late MCI (LMCI) subjects. Detailed inclusion criteria for the diagnostic categories can be found at the ADNI website . Demographics and neuropsychological profiles of the different diagnostic groups are summarized in Data were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (http://adni.loni.usc.edu/methods/mri-tool/mri-pre-processing/). FDG- and AV45-PET data were acquired on multiple instruments of varying resolution and following different platform-specific acquisition protocols. Similar to the MRI data, PET data in ADNI were also subject to standardized image preprocessing correction steps, with the aim of increasing data uniformity across the multicenter acquisitions . Imaging data were processed by using statistical parametric mapping  and the VBM8 toolbox  implemented in MATLAB R2013b  as previously described in Grothe et al. /sres, j; with xij being the ith subject's raw value for region j; eij being the value expected for region j in the control group for the ith subject's age, gender, and education; and sres, j being the standard deviation of the residuals for region j in controls.ADNI-GO/2 MRI, FDG- and AV45-PET data were downloaded from the ADNI image archive. ADNI-GO/2 MRI data were acquired on multiple 3T MRI scanners using scanner-specific T1-weighted sagittal 3D MP-RAGE/IR-SPGR sequences. To increase signal uniformity across the multicenter scanner platforms, original T1 acquisitions underwent standardized image preprocessing correction steps  and all three imaging modalities were entered. Initially, we took all 108 cortical and subcortical regions included in the Harvard-Oxford atlas  to increase model stability. From the final model we set a probability threshold of Pavg > 0.5 for selecting the edges, with the notion that a specific edge was considered to be present if it existed in at least half of all model iterations  using data of the right hemisphere to validate the results and (ii) using the traditional approach of constructing correlation networks based on the Pearson correlation coefficient.i∈N and all other nodes, i and j, gi↔j defines the shortest path, and ωuv defines the distance between two nodes. Here, the distance matrix was defined as Ω = 1−abs(Θ), that is one minus the absolute pair-wise partial correlation as derived from the GGMs or the absolute Pearson coefficient, respectively ), where i, and where i /(L/Lrand), with S≫1 in small-world networks  and Tukey's honest significant difference tests.To assess group differences of the estimated graph structure we calculated the three graph-theoretical measures that are most commonly reported in the literature; clustering coefficient, characteristic path length, and their ratio, the small-world coefficient. The path length quantifies the distance of connections between two nodes along the shortest path. The weighted characteristic path length is the average minimum distance between a node P > 0.5 , corresponding to a network density of 46% of the total number of possible edges.The conditional dependency matrix obtained using the GGM approach for all region of the left hemisphere is given in For intra-modal associations, i.e., within the same imaging modality, brain regions directly adjacent to each other formed smaller clusters of high partial correlation around the main diagonal –5. When When estimating separate models for each diagnostic group based on the multimodal data, graph structures derived from Pearson and partial correlation matrices –8 both ddf = 215, F ≥ 4, p < 0.01, η2 ≥ 0.055. Detailed results are provided in P-values for Tukey's honest significant difference tests are provided in We observed a biphasic trajectory of the graph measures. This means that the clustering coefficient and small world coefficient initially increases when comparing early MCI and CN participants . When Alwithin imaging modalities. We expected adjacent brain regions to form clusters with high inter-cluster similarity for amyloid-β deposition  might be explained by the asymmetry of the brain and the lateralization reported for Alzheimer's disease in the literature  . Processed imaging data and extracted regional mean values are available from the corresponding authors upon request. The R package BDgraph can be downloaded from CRAN (https://cran.r-project.org/web/packages/BDgraph) or GitHub (https://github.com/cran/BDgraph).MRI and PET data being used in this study can be retrieved from ADNI (The studies involving human participants were reviewed and approved by ADNI internal review board. The patients/participants provided their written informed consent to participate in this study.MD, RM, TK, and ST designed the study. MG and MD preprocessed the imaging data. MD and RM conducted the statistical analyses. MG, TK, and ST aided in interpreting the results. MD drafted the first version of the manuscript. All authors revised the manuscript and contributed to the final 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.


Tourette syndrome (TS) is a developmental neuropsychiatric disorder with onset during childhood. Because of its complex spectrum of phenotypes, the underlying pathophysiology of TS is still unclear. Resting-state functional magnetic resonance imaging demonstrated aberrant spontaneous neural synchronization in conventional frequency band (0.01–0.08 Hz) in TS. No published studies have reported abnormalities of local synchronization across different frequency bands. We estimated the alterations of local synchronization across five bands ranging from 0 to 0.25 Hz. Seventy-nine children with TS and 63 age-, sex-, and handedness-matched healthy children were recruited. Frequency-specific regional homogeneity (ReHo) and independent component analysis were used to identify functional alterations between TS and healthy children. TS patients showed significantly increased ReHo in the left precentral gyrus and decreased ReHo in the right operculum. Abnormal ReHo alterations of the superior frontal gyrus, superior parietal gyrus, anterior cingulate gyrus, putamen, superior temporal gyrus, and operculum were observed in different frequency bands. TS patients showed increased connectivity of the right superior frontal gyrus within the left executive control network. In addition, a significantly negative correlation was found between Yale Global Tic Severity Scale (YGTSS) vocal score and ReHo values of the right operculum in the highest frequency bands (0.198–0.25 Hz), while a significant positive correlation was found between YGTSS motor score and altered connectivity of the right superior frontal gyrus. The present study revealed frequency-specific abnormal alterations of ReHo in the whole brain and altered connectivity within the executive control network of TS children. Its neural importance and clinical practicability require further investigation. Tourette syndrome (TS) is a developmental neuropsychiatric disorder with an onset during childhood, defined by the co-occurrence of motor and phonic tics lasting at least 12 months. Tics typically show a waxing and waning pattern of severity, intensity, and frequency , which mAdvanced neuroimaging methods have been used to investigate the neural basis of TS over the last few decades. Many neuroimaging studies using structural magnetic resonance imaging (MRI), positron emission tomography (PET), and diffusion tensor imaging techniques have reported abnormalities in the basal ganglia and associated thalamic and frontal cortical regions , supportn = 21) showed decreased ReHo in the right cerebellum that was positively correlated with disease duration  has the advantage of measuring spontaneous neural activity such as regional homogeneity (ReHo) in the TS population , 11. A pduration . In contduration . Frequenduration . Thus, aTo discover frequency-specific functional alterations that might have an important role in the pathophysiology of TS, we used RS-fMRI in a frequency-specific manner  to compare TS patients with healthy controls (HC) , 15. IndNinety-four children with TS were recruited from outpatient clinics of the Second Affiliated Hospital, Zhejiang University School of Medicine. All patients met the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, text revision (DSM-IV-TR) criteria. Sixty-three age-, sex-, and handedness-matched healthy children without any neurological or psychiatric disorders were recruited as HC. Participants were excluded if they had (i) any neurological (other than tics) or psychiatric diseases (other than ADHD and OCD), (ii) structural abnormalities on visual inspection of structural imaging, and (iii) head motion exceeding 3 mm in translation or 3° rotation in any direction. After head-motion control, fifteen patients were excluded. Seventy-nine TS patients were eligible to take part in the current study. All clinical evaluations were performed on the day of acquisition of MRI scans by an experienced pediatric neurologist . The YalMRI images were acquired on a 3.0-Tesla MRI scanner  at the Center for Cognition and Brain Disorders, Hangzhou Normal University. Foam pads were used to minimize head motion for all subjects. A gradient-recalled echo planar imaging sequence  was used to obtain functional images. Forty-three axial slices  were acquired. Participants were instructed to rest with their eyes closed, not to think of anything in particular, and not to fall asleep. Three-dimensional T1-weighted images were obtained in the sagittal orientation using a magnetization-prepared rapid acquisition gradient-echo sequence . After each scanning session, the responsiveness of the subjects was determined by asking whether they had fallen asleep during the scan.http://www.fil.ion.ucl.ac.uk/spm) software and the Data Processing & Analysis for Brain Imaging (DPABI) toolbox (http://rfmri.org/dpabi) were used for preprocessing  was used to calculate spatially independent components (ICs). Dimension estimation of data from both groups was conducted using the minimum description length (MDL) criterion to determine the number of ICs  for further analysis. The brain networks included basal ganglia, sensorimotor, and the left and right executive control networks  was 43. These components were selected based on the largest spatial correlation with a specific resting-state network template . The peak ReHo values of the clusters surviving from the group main effect were extracted and then entered SPSS software for two-sample t-tests in each frequency bandTwo-way repeated-measures analysis of variance (ANOVA) was performed to examine the effects of group and frequency band on the ReHo maps. Group (TS and HC) served as a between-subject factor and frequency band  served as a within-subject factor. An F-map of the “frequency by group” interaction effect and an F-map of the group main effect were obtained. Multiple-comparison correction was conducted based on Gaussian random field (GRF) theory .To explore connectivity differences between the two groups, two sample p = 0.599), sex (p = 0.066), and handedness (p = 0.370) between the two groups. The detailed demographic variables and clinical characteristics of participants are shown in There were no significant differences in age  (The two-sample l lobes) .The ReHo value of the right operculum  showed significant negative correlations with vocal scores of YGTSS in the highest frequency bands (0.198–0.25 Hz)  within the left executive control network . Task-maintenance processes may be affected resulting in unwanted breakthroughs of normally suppressed behaviors, such as tics. However, the hyperactive frontal-parietal network  motivated initiate and adjust change in task control, again leading to unwanted movements and vocalizations . In prevIn addition to local synchronization, the present study used ICA to detect connectivity differences within the basal ganglia, sensorimotor, and executive control networks. We found increased connectivity of the right superior frontal gyrus within the left executive control network. The executive control network shares major nodes with the frontal-parietal network, including the dorsolateral prefrontal cortex and posterior parietal cortex. It is involved in the control of higher-order cognitive neural functions such as attention, planning, decision-making, and working memory . Recent To the best of our knowledge, this is the first study to examine abnormalities of regional synchronization in a relatively large and typical pediatric TS population using the frequency specificity of ReHo. Several studies demonstrated the frequency specificity of ReHo changes in neurologic and psychiatric disorders, such as paroxysmal kinesigenic dyskinesia , schizopThe significant negative correlation between ReHo and YGTSS scores was found only in the highest frequency bands (0.198–0.25 Hz) , Table 5In summary, the present RS-fMRI study revealed frequency-specific abnormal regional homogeneity and altered connectivity in children with TS. The regions of frequency-specific abnormalities included the frontal–parietal network, ACC, and putamen, as well as an altered connectivity region in the executive control network. These brain regions are involved in multiple cognitive dysfunctions characteristic of TS. All these aberrant regions might be direct treatment targets or indirect targets for brain stimulation, especially those regions with frequency-specific abnormalities, which might be sensitive to specific stimulation frequencies for TMS. These targets should be tested and verified in TS populations in the future.This study had several limitations. The drugs used for TS treatment may affect brain function. The dose and duration of drug information were not recorded completely in the current study. The variability of the doses and types of drugs used makes it difficult to analyze the contribution of each drug on ReHo. Therefore, drug-naïve status and type of medication used should be taken into consideration in the future.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Center for Cognition and Brain Disorders, Hangzhou Normal University, China. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.Y-TL: design the study, statistical analysis, and writing of the first draft. X-LL: fMRI data processing and statistical analysis. YW: neurological evaluation and psychiatric evaluation. G-JJ: fMRI data acquisition. Y-FZ: revised the article and developed the research concept. JW: fMRI data processing and statistical analysis, revised the article, and developed the research concept. J-HF: research project conception and organization, neurological evaluation, revised the article, and developed the research concept. 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.