The aim of this study is to investigate the long‐term effects on jaw and femur bone induced by oncologic doses of zoledronic acid in a young rat model.Six 12‐week‐old male Wistar rats received zoledronic acid (0.6 mg/kg) and six control rats received saline solution in the same volume. Compounds were administered intraperitoneally in five doses every 28 days. Euthanasia was performed 150 days after therapy onset. After animal sacrifice, their mandibles and femurs were scanned ex vivo using a high‐resolution (14 μm) micro‐computed tomography. Morphometric bone parameters were calculated using CT‐Analyzer  between the first and second mandibular molars and in the distal femur metaphysis and epiphysis.p < .05) increased bone quantity , bone density , and osteosclerosis of the trabecular bone  in all anatomical sites. Bone remodeling suppression due to zoledronic acid treatment was more pronounced (p < .05) in the femoral metaphysis relative to the mandible and epiphysis. The exploratory linear discriminant analysis showed that for the mandible, it was mainly the bone quantity‐related morphometric indices (BV/TV and Tb.Th), while for the femoral epiphysis and metaphysis, it was bone structure‐related (Tb.Pf and Tb.N), which are of primary importance to study the treatment effect.The treatment group as compared to the controls showed a significantly (High doses of bisphosphonates can differently affect the bone quantity, density, and structure in long bones and jawbones. In the metaphysis, bone changes were primarily concentrated in the region of the growth plate. Future studies may consider the use of bone morphometric indices to evaluate the effect of bisphosphonates. IntraveUncommon late site‐specific side effects, such as atypical fracture of the femur and osteonecrosis of the jaw, have been related to long‐term therapies with high‐dose bisphosphonates in adults  is a validated method for the high‐resolution evaluation of tridimensional bone structure, presenting high accuracy compared to histomorphometry  and all data are reported according to the ARRIVE  criteria  scan protocol. Each sample was placed in a 1.5 ml Eppendorf tube with saline solution and scanned with a SkyScan1174 micro‐CT . Scanning parameters were set at 50 kVp, 800 μA, frame averaging of 6° and 180° rotation with an angular step of 0.8°. A 0.5‐mm‐thick aluminum filter was used to reduce noise artifacts and to minimize beam hardening effects that could affect the further analysis. Hydroxyapatite phantoms of 0.25 and 0.75 g/cm3 (Bruker) were used and scanned according to the scan protocol to perform a BMD calibration with respect to the attenuation values. The cross‐sectional images were reconstructed from the projection images in NRecon (Bruker). After reconstruction, micro‐CT images were registered using a MeVisLab framework  to spatially align all scans to ensure a uniform comparison of anatomical structures , total porosity percentage (Po[tot] in%), specific bone surface (BS/TV in %), and trabecular thickness (Tb.Th in mm); (2) bone structure: trabecular number (Tb.N in 1/mm), trabecular separation (Tb.Sp in mm), connectivity density (Conn.Dn in 1/mm3), degree of anisotropy (DA), trabecular pattern factor (Tb.Pf in 1/mm), and structure model index (SMI) as well as (3) bone density: tissue mineral density (TMD in mg HA/cm3) and bone mineral density (BMD in mg HA/cm3).Morphometric bone indices were operator‐independent calculated and blinded for treatment regimen, based on the segmented trabecular bone structure and were grouped according to terms clinically used for bone quality evaluation  and group . Post hoc Bonferroni‐corrected tests were used to explore significant interaction effects. Mean and standard deviations of each parameter were reported. An exploratory linear discriminant analysis for each anatomical site was performed to identify the morphometric parameters that best predict group membership. The strength of the canonical correlation (cc) was used to indicate the discriminatory power of each morphometric parameter for group classification. Leave‐one‐out cross‐validation was used to assess model prediction performance and how the results of the LDA will generalize to an independent data set. Statistical analyses were performed in SPSS  at a significant level of .05.The minimum required sample size was calculated using the effect size of a previous comparison study between control and zoledronic acid‐treated rats with a similar design . Zoledronic acid‐treated rats showed an overall larger bone quantity  related to thicker (larger Tb.Th) and less complex trabeculae . The trabecular bone structure after zoledronic acid treatment showed typical osteosclerotic characteristics marked by a smaller number of trabeculae  and more plate‐like structure (lower SMI), explained by smaller trabecular spaces , more enclosed cavities , and heterogeneous density distribution . No significant changes in BMD were observed.Administration of zoledronic acid caused a significant overall change in bone quality in all bones  in comparison to femoral metaphysis and epiphysis  and in the mandible (an increase of 30% and 23%) compared to the control group.3.2.4All bone specimens of the three anatomical sites were correctly classified in the corresponding treatment group. Cross‐validation showed a 100% prediction accuracy for the metaphysis, 83.3% for the mandible and 72.7 for the epiphysis. The distinctive importance of the morphometric parameters was specific for each anatomical site. For the mandible, BV/TV (cc = 0.88), Tb.Th (cc = 0.80), Tb.Sp (cc = −0.65), and DA (cc = 0.64) were more important for the performance of the given discriminant model, while for the metaphysis Tb.Pf (cc = −0.79), SMI (cc = −0.66), Tb.N (cc = 0.63) were the most important distinctive parameters. For the epiphysis, the cc parameters were smaller Tb.Pf (cc = 0.60), BV/TV (cc = 0.58), and Tb.N (cc = 0.57) in comparison to the metaphysis and mandible.4Over the past few years, bisphosphonate indications for the treatment of several chronic metabolic bone conditions have emerged, being increasingly used in young populations (Simm et al., Previous studies in adult patients have demonstrated that weight‐bearing sites, such as tibia, are more susceptive to bisphosphonate antiresorptive effect (Burghardt et al., Preclinical studies have demonstrated that bisphosphonates have A high atypical fracture of the femur and induce higher remodeling suppression in the jaws compared to other bones (Wen et al., In our investigation overall, in the zoledronic acid group, the mandible also presented denser bone with smaller marrow spaces compared to femur metaphysis. This result is in accordance with Vermeer et al. , who alsEvidence shows that lower doses of bisphosphonates improve bone volume fraction and increase the trabecular number, while higher doses increase the trabecular thickness (Gou et al., There are several bone disorders affecting young patients in which bisphosphonate treatment has been recommended, including osteogenesis imperfecta, idiopathic juvenile osteoporosis, secondary osteoporosis, fibrous dysplasia, and skeletal neoplasms (Simm et al., The present study showed that high‐dose, long‐term zoledronic acid treatment had a site‐specific effect on the mandible and femur during bone growth. Clinically, however, osteonecrosis only occurs in the jaw. The difference between long bones and jawbones, besides their mode of ossification (Simm et al., This study has some limitations that need to be considered when interpreting its findings. First, the analysis was limited to ex vivo micro‐CT imaging. This meant that the process of bone remodeling throughout rat development could not be studied. Longitudinal dynamic bone morphometry may give more insight into bone remodeling changes due to bisphosphonate treatment. Second, our experimental model received one bisphosphonate dose scheme. Using different drug doses and intervals may add information to the discussion of the optimal bisphosphonate regimen considering its risks and benefits. Third, despite the different bone composition between the mandibula and the maxilla, the bone morphometric parameters of the maxilla were not analyzed in this study. Due to the fact that there are too many additional differences between the maxilla and the studied bone types that not be corrected for in the present study. Previous research has shown that the bone turnover in the mandible is similar to that in the femur and significantly lower than that in the mandible (Ristow et al., In conclusion, we demonstrated differential site‐specific effects of zoledronic acid in the long bones and jawbones during bone growth in a rat model. In the femur distal metaphysis, the changes are primarily concentrated in the region of the growth plate. Future studies may consider the use of bone morphometric indices to evaluate the effect of bisphosphonates.All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Mariana Quirino Silveira Soares, Jeroen Van Dessel, Gustavo Zanna Ferreira, Paulo Sérgio da Silva Santos, Laura Ferreira Pinheiro Nicolielo, and Marco Antônio Húngaro Duarte. The first draft of the manuscript was written by Mariana Quirino Silveira Soares and all authors commented on previous versions of the manuscript. This research was supervised by Reinhilde Jacobs and Izabel Regina Fischer Rubira‐Bullen. All authors read and approved the final manuscript.The authors declare no conflict of interest.


Chemoresistance in patients with glioblastoma multiforme (GBM) is a common reason hindering the success of treatment. Recently, ferroptosis has been reported to be associated with chemoresistance in different types of cancer, while the role of ferroptosis‐related genes in GBM have not been fully elucidated. This study aimed to demonstrate the roles and mechanism of ferroptosis‐related genes in chemoresistance and metastasis of GBM. First, two candidate genes, squalene epoxidase (SQLE) and FANCD2, were identified to be associated with ferroptosis‐related chemoresistance in GBM from three temozolomide (TMZ) therapeutic datasets and one ferroptosis‐related gene dataset. Then, comprehensive bio‐informatics data from different databases testified that SQLE was significantly downregulated both in GBM tissue and cells and displayed a better prognosis in GBM. Clinical data identified lower expression of SQLE was significantly associated with WHO grade and 1p/19q codeletion. Moreover, through in vitro experiments, SQLE was confirmed to suppress ERK‐mediated TMZ chemoresistance and metastasis of GBM cells. The KEGG analysis of SQLE‐associated co‐expressed genes indicated SQLE was potentially involved in the cell cycle. Furthermore, SQLE was found to have the most significant correlations with tumor‐infiltrating lymphocytes and immunomodulators. These findings highlighted that SQLE could be a potential target and a biomarker for therapy and prognosis of patients with GBM. Chemoresistance in glioblastoma multiforme (GBM) is a common reason for tumor recurrence and ferroptosis has been reported to correlate with chemoresistance in cancers. Here, we identified a ferroptosis‐related gene SQLE that was significantly associated with the prognosis of patients with GBM. Moreover, SQLE was confirmed to suppress ERK‐mediated TMZ chemoresistance and metastasis of GBM cells. Our study proposed a novel target for the treatment of chemoresistant in GBM. One ferroptosis‐related gene, SQLE, was identified to have an impact on chemotherapy response in GBM. Furthermore, lower SQLE expression was recognized in GBM and was correlated with poor prognosis. The expression levels of SQLE might be a positive correlation with TMZ activity in glioma cells. In addition, in vitro studies, we tested that SQLE was lowly expressed in TMZ‐resistant GBM cells and involved in ERK‐mediated TMZ resistance of GBM cells. Overexpression of SQLE could significantly inhibited the migration and invasion of GBM cells. Moreover, the KEGG analysis showed SQLE was potentially involved in the cell cycle. Furthermore, results of tumor‐immune system interactions and drug bank database (TISIDB) demonstrated that SQLE had the most significant correlations with tumor‐infiltrating lymphocytes and immunomodulators.22.1The functions of ferroptosis‐related chemoresistance of GBM were investigated using several public databases Table . Three TThe expression levels of SQLE in GBM were explored through the GlioVis, The Human Protein Atlas (THPA),2.2The TMZ‐resistant glioma cell lines (T98g‐R and U118MG‐R) and their parental cell lines (T98G and U118MG) were cultured as previously described.2.32 at 37°C. After two weeks, cell colonies were fixed with 4% paraformaldehyde (30 min), and stained with 0.1% crystal violet (20 min). Colonies with more than 50 cells will be counted.The T98G‐R and U118MG‐R cells were sufficiently trypsinized and suspended in a complete medium. Then, cells were seeded in 6‐well plates (500 cells/well) and cultured under 5% CO2.44/chamber), medium with 20% FBS was added into the lower chamber. After 24 h, the cells were fixed  and stained . Under the microscope, each group randomly selected 10 high‐power fields of view for counting and statistics.For invasion assays, the upper chamber was placed into the 24‐well lower chamber. The upper membranes were coated with 40 μl of matrigel  in advance. The cells were resuspended in FBS‐free medium and were seeded in the upper chamber  and blank plasmids (G0206627‐2) were constructed by Genepharma . The ERK overexpression plasmid was constructed by WZ Biosciences Inc . T98G‐R and U118MG‐R cells were transfected with these plasmids using Lipofectamine 3000 reagent (Invitrogen).2.7t‐test was applied to compare differential expression between two groups. The Kaplan–Meier analysis was used to analyze GBM prognosis. The Kruskal–Wallis rank test or the Mann–Whitney U test was used to analyze the associations between SQLE expression and clinicopathologic characteristics. Pearson's correlation coefficient was used to analyze the correlations between genes. All the experiments were conducted in triplicate with mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 were defined as statistically significant.Statistical analyses were performed with SPSS 19.0  and GraphPad Prism 8  software. Normality was analyzed using the Shapiro–Wilk test. Student's 33.1p < 0.05 and |logFC| > 2, we identified 5,475 genes in GSE47809, 3961 genes in GSE65363 and 1857 genes in GSE80729. Next, 156 genes were further identified to be significantly co‐differentially expressed among the aforementioned three datasets by the Venn analysis . Furthermore, we explored the correlation between SQLE expression and clinicopathological features in glioma, the results showed that SQLE expression was significantly associated with WHO grade and 1p/19q co‐deletion, and was not related to age, gender, IDH status, and MGMT methylation status  and their parental cells (T98G and U118MG cells). The results showed that SQLE expression decreased significantly in TMZ‐resistant glioma cell lines Figure , suggestResearches have shown that MAPK/ERK signaling pathway plays a pivotal role in the development and chemoresistance of GBM.3.4Several studies demonstrated that ERK was involved in the invasion and metastasis of the GBM,We further explored whether SQLE affects the migration and invasion ability of GBM cells through ERK pathway. We found that compared with either of the individual treatment groups, the migration and invasion ability of TMZ‐resistant GBM cells transfected with both SQLE overexpression plasmid and KO947 were suppressed most Figure . Besides3.5p < 0.01 , monocyte cells , myeloid‐derived suppressor cells , and activated dendritic cells  , CD86 , C10orf54 , and CD48  , CD244 , HAVCR2 , and PDCD1LG2  , HLA–DRA , HLA–DMA , and HLA–DOA   Figure . Further) Figure ; those i) Figure ; and the) Figure . Based o4There is an urgency to find effective biomarkers and improve treatment strategies based on the fact that patients with GBM suffer from poor prognosis and chemoresistence. A recent study showed that the ferroptosis gene signature could predict immunotherapy and prognosis in patients with glioma, indicating that ferroptosis plays an important role in the progression of glioma.SQLE is the key rate‐limiting enzyme in cholesterol biosynthesis and a target of fungicides and increasing interest in human health and disease.GBM is the most common type of grade IV gliomas.Nevertheless, our research still has some limitations. The expression and prognosis data of SQLE were mainly obtained from TCGA, which mainly included white and black, which made our conclusions suitable for limited ethnic populations. Besides, several genes are involved in ferroptosis related signaling pathways, such as GPX4, Nrf2, SOD, MDA, and so on.To sum up, this is the first study that SQLE is associated with the ferroptosis‐related chemoresistance in GBM and the low expression of SQLE was related with poor prognosis and low‐immune infiltration. Furthermore, SQLE was related to ERK‐mediated TMZ resistance of GBM cells. Overexpression of SQLE could inhibited the migration and invasion ability of GBM cells. Therefore, SQLE could be used as a potential therapeutic target and as a promising prognosis biomarker for patients with GBM.All the authors made substantive intellectual contributions to this study to qualify as authors. Kuan Hu and Juanni Li conceived the design of the study. Lei Yao and Xiaofang Zhang performed the study, collected the data and contributed to the design of the study. Lei Zhou and Lei Yao edited the manuscript. All the authors read and approved the final manuscript.This study is supported by grants from the National Natural Science Foundation of China , Outstanding Postdoctoral Innovative Talents Foundation (2021RC2022), Youth Science Foundation of Xiangya Hospital (2020Q07)The authors declare no conflict of interest.Appendix S1Click here for additional data file.Figure S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.


High-throughput metabolomics was conducted with the patients’ serum samples using mass spectrometry. After controlling for CKD severity and other clinical hypertension risk factors, we identified ten metabolites that have significant associations with blood pressure. The quantitative importance of these metabolites was verified in a fully connected neural network model. Of the ten metabolites, seven have not previously been associated with blood pressure. The metabolites that had the strongest positive association with blood pressure were aspartylglycosamine (p = 4.58 × 10−5), fructose-1,6-diphosphate (p = 1.19 × 10−4) and N-Acetylserine (p = 3.27 × 10−4). Three metabolites that were negatively associated with blood pressure  have been reported previously to have beneficial effects on hypertension. These results suggest that intake of metabolites as supplements may help to control blood pressure in CKD patients. Blood pressure is one of the most basic health screenings and it has a complex relationship with chronic kidney disease (CKD). Controlling blood pressure for CKD patients is crucial for curbing kidney function decline and reducing the risk of cardiovascular disease. Two independent CKD cohorts, including matched controls (discovery  Hypertension is the leading risk factor for many diseases, including cardiovascular disease, stroke, kidney disease, etc.  One of tMetabolites are organic compounds, produced intermediately during metabolism. They are used for chemical reactions in cells. Metabolites have been thoroughly studied in CKD. It has been suggested that metabolites have strong clinical value for CKD . StudiesMetabolites have been studied closely with CKD and blood pressure independently, but never in conjunction. In this study, using an approach combining traditional statistical methods and deep learning methods, we show that, within the confinement of CKD, certain metabolites are directly associated with blood pressure after adjusting for CKD severity. p = 0.06) and the combined dataset (p = 0.07), but not in the validation cohort (p = 0.6). Sex was not associated with blood pressure. Age, weight, BMI and eGFR were found to be significantly associated with blood pressure in the discovery, validation and combined cohorts. In the combined cohort, weight and BMI had the largest effects on blood pressure . These results indicate that increases in age, weight and BMI and decline in kidney function are associated with increase in blood pressure, which is consistent with previous findings. Based on regression results, in the combined cohort, the clinical characteristics together explained 20% variance in blood pressure (adjusted R2 = 0.20).Two cohorts with 1376 subjects  were used for this study. The basic clinical characteristics are summarized in All the subjects’ serum samples went through mass spectrometry to identify metabolites. A total of 25,107 features were identified. To remove collinearity within the features and reduce the dimensionality of the analysis, we performed feature reduction using the correlation clustering algorithm , after wThe relationships between these 1755 selected features and blood pressure were evaluated using a linear regression model while adjusting for CKD stage in the discovery cohort, then in the validation cohort. In both cohorts, 31 features were found to be significantly associated with blood pressure, and the exact metabolites for these 31 features were identified. Final regression models were carried out by combining data from both cohorts . In the 2 = 0.1342). By combining clinical characteristics, the total explainable variance of blood pressure increased to 23.29% . This model had a prediction accuracy of 46%, which was better than random chance  but far from being suitable for application in a clinical setting. By adding the additional ten metabolites, the model’s accuracy increased to 52% . Hypertension is both a cause and a consequence of CKD, making controlling blood pressure a vital strategy to slow down CKD progression [It is estimated that around 20% of the world’s adults have hypertension. Hypertension is closely intertwined with many diseases, including CKD, which has an estimated global burden of 10% . Aspartylglycosamine was firstly identified as a biomarker of hypertension under CKD and also as a biomarker of a congenital disorder of deglycosylation [p = 1.19 × 10−4). As a glycolytic intermediate, fructose 1,6-diphosphate treatment mitigated ischemic acute renal failure [We designed a study to examine the relationship between metabolites and blood pressure within the context of CKD. Two large cohorts  of 1376 subjects were recruited. Metabolites were detected in the patients’ serum samples using mass spectrometry. Through a series of statistical analyses, we identified ten metabolites associated with blood pressure after adjusting for CKD severity. The importance of these ten metabolites was further validated through statistical analysis of Rsylation ,20. Ther failure  and prev failure . Even th failure  and has  failure , which mp = 6.39 × 10−3), which had similar trends with a previous study in cardiovascular disease [p = 0.01). Although no direct association with blood pressure has been found, dodecanedioic acid was found to help maintain blood sugar levels [Phosphocreatine was negatively associated with blood pressure . A previous study showed trends of 3-methyladenine levels consistent with our study of hypertension. After treatment with 3-methyladenine, vascular smooth muscle cells were sensitive to senescence that contributed to hypertension, indicating 3-methyladenine as a potential risk factor for hypertension [N-acetylneuraminic acid was negatively associated with blood pressure. However, a previous study showed that N-acetylneuraminic acid was positively associated with hypertension in patients with cardiovascular disease, which may be a result of different pathological states [p = 0.04), which is more concordant with the second study. Additionally, in the present study, we identified that a backbone of mammalian cell membranes, sphingosine phosphate, was negatively associated with blood pressure. A number of studies have confirmed the protective effects of sphingosine phosphate in hypertension and blood pressure regulation, indicating that the modulation of sphingosine phosphate signaling is a potential therapeutic target of hypertension under CKD ,34,35,36rtension . Here, wl states . Our stul states . Howeverl states . In our Our study contained two large cohorts of CKD patients and matched controls. All participants were ethnically Chinese, and all patients provided written informed consent. The first cohort contained 824 subjects  who were recruited between February 2011 and 2013 from the Affiliated Hospital of Shaanxi Institute of Traditional Chinese Medicine. The second cohort contained 552 subjects  who were recruited between 2013 and 2016 from Xi’an No. 4 Hospital and Baoji Central Hospital. The four-variable equation of the Modification of Diet in Renal Disease (MDRD) Study was used to estimate GFR (eGFR) . SubjectBlood pressure was classified into five groups based on conventional definition: normal (systolic < 120 and diastolic < 80), elevated (systolic: 120–129 and diastolic < 80), high stage 1 (systolic: 130–139 and diastolic: 80–89), high stage 2 (systolic: 140–180 or diastolic: 90–120) and hypertensive (systolic > 180 or diastolic > 120). Blood pressure was subsequently coded numerically as 1 to 5 for further analysis. Serum samples were collected from all participants. Serum samples were obtained after overnight fasting and sera were separated and stored at −80 °C for biochemical analysis. Blood biochemistry was determined by the clinical laboratory. The metabolomic procedure included sample preparation, metabolite separation and detection. Data preprocessing and statistical analysis for metabolite identification was performed following our previously described protocol [Mass spectrometry was performed with a Waters XevoTM G2 QTof MS. The scan range was from 50 to 1200 m/z. For positive and negative ESI modes, the capillary and cone voltages were set at 2.5 kV and 45 V, respectively. The desolvation gas was set at 550 °C with a desolvation gas rate of 900 L/h. The source temperature was set at 120 °C, and the cone gas rate was set as 50 L/h. The data were obtained in centroid mode. The LockSpray frequency was set as 10 s, and the data were averaged over 10 scans. The data acquisition rate was set to 0.1 s with a 0.1 s interscan delay. During analysis, centroid data were collected with a scan time of 0.1 s and an interscan delay of 0.02 s. Leucine–enkephalin was used as the lockmass at the level of 300 ng/mL with a flow rate of 5 μL/min.To obtain a pooled quality control sample, 50 µL of all the samples were pooled, and 10 ions, including m/z 161.9852, 391.2812, 381.2846, 764.5300, 441.1958, 820.8169, 208.1360, 133.0861, 486.2534 and 429.2741, were extracted for assessment. The continuous analyses of 6 replicates of quality control samples were used to measure injection precision. RSD% of retention times and peak areas were also measured using quality control samples. The sample preparation repeatability was calculated by 6 parallel samples, and the method repeatability of RSD% of retention times and peak areas of 10 ions was measured from quality control samples. The above-mentioned procedure was performed every day.Algorithm 1 Correlation Clustering AlgorithmV, E+, E−    H ← ∅  while V ≠ ∅ do   C ← ∅   V’ ← ∅   Pick a random pivot i ∈ V   C ← {i}   for j ∈ V and j ≠ i do    if j, i ∈ E+ then     Add j to C    end if     if j, i ∈ E− then     Add j to V’    end if   end for   V ← V’   Add C to H  end while  return HRequire: Overall, 25,107 features were identified through mass spectrometry. Each feature was described by its retention time and molecular weight. Many of the features were highly correlated. We conducted feature reduction using a correlation clustering algorithm (Algorithm 1) . The algAlgorithm 2 Pearson Correlation Threshold Checki, j, t  P ← pearson  Q ← |P|  return Q > tRequire: In the discovery cohort, linear regression was carried out to evaluate the relationship between blood pressure and metabolites. To account for the potential correlation between metabolites and CKD stages, an interaction term between feature and CKD stage was added to the regression model. Statistically significant features recurrent in the model of the validation cohort were retained. A final regression model was conducted combining samples from both cohorts. The Benjamini–Hochberg method was used as the multiple test correction method.A feature importance algorithm  was usedE fragment, literature comparisons and database searches, including the Human Metabolome Database , KEGG , METLIN , MassBank  and Chemspider . Additionally, some metabolites were confirmed by comparison with available reference standards under the same UPLC–HDMS condition.The final list of blood pressure-associated metabolites was identified and annotated using exact molecular weights, m/z element composition using MassLynx i-FIT software , MS, MSThis study was approved by the Ethical Committee of Shaanxi Traditional Chinese Medicine Hospital (permit number: SXSY-235610), and written informed consent was received from all participants before inclusion in the study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.By conducting a large-scale high-throughput metabolomics study, we identified ten metabolites that were associated with blood pressure after adjusting for the severity of CKD and other hypertension risk factors. With clinical characteristics and metabolites, we explained 23.29% of the variance in blood pressure. A deep learning model that utilized both clinical characteristics and metabolites reached an accuracy of 52%. Although better than random chance, these results are not clinically meaningful and further demonstrate that hypertension is a complex disease with many potential risk factors. The identified metabolites potentially hint at alternative metabolomics-based strategies for controlling hypertension in CKD patients.


The effectiveness of lipid‐lowering therapy (LLT) is affected by both intensity and adherence. This study evaluated the associations of LLT intensity, adherence, and the combination of these 2 aspects of LLT management with the risk of major adverse cardiovascular events (MACE) in people with coronary heart disease.This is an observational study of all adults who suffered a myocardial infarction or had coronary revascularization during 2012 to 2018 and initiated LLT in Stockholm, Sweden. Study exposures were LLT adherence (proportion of days covered), LLT intensity (expected reduction of low‐density lipoprotein cholesterol), and the combined measure of adherence and intensity. At each LLT fill, adherence and intensity during the previous 12 months were calculated. The primary outcomes were MACE ; secondary outcomes were low‐density lipoprotein cholesterol goal attainment and individual components of MACE. We studied 20 490 patients aged 68±11 years, 75% men, mean follow‐up 2.6±1.1 years. Every 10% increase in 1‐year adherence, intensity, or adherence‐adjusted intensity was associated with a lower risk of MACE  and higher odds of attaining low‐density lipoprotein cholesterol goals . Among patients with good adherence (≥80%), the risk of MACE was similar with low‐moderate and high‐intensity LLT despite differences in the low‐density lipoprotein cholesterol goal attainment with the treatment intensities. Discontinuation ≥1 year increased the risk markedly .In routine care, good adherence to LLT was associated with the greatest benefit for patients with coronary heart disease. Strategies that improve adherence and use of intensive therapies could substantially reduce cardiovascular risk. LLTlipid‐lowering therapyMACEmajor adverse cardiovascular eventsPDCproportion of days coveredBoth adherence and treatment intensity can alter the effectiveness of lipid‐lowering therapy (LLT) in routine clinical practice; suboptimal LLT management may affect cardiovascular disease recurrence and low‐density lipoprotein cholesterol (LDL‐C) goal attainment.This study evaluated a combined measure of adherence and treatment intensity using a roll‐out design, and we assessed adherence at each LLT fill. This minimizes the immortal‐time bias of evaluating treatment adherence within fixed periods.We controlled for LDL‐C levels and the frequency of LDL‐C testing, which are important time‐dependent confounders, because regular LDL‐C monitoring may indicate health‐seeker or compliant behaviors and motivate treatment decisions.Optimal LLT use had a significant prognostic benefit, regardless of treatment intensity, in post–myocardial infarction or revascularized patients with coronary heart disease.Good adherence to LLT was more important than LLT intensity or achieved LDL‐C levels for the reduction of cardiovascular risk in patients with coronary heart disease.The highest cardiovascular risk was observed among those who discontinued LLT. Strategies that improve adherence and greater use of intensive therapies could substantially reduce cardiovascular risk in secondary prevention.Patients with established coronary heart disease (CHD) require targeted risk management strategies, including effective lipid‐lowering therapy (LLT) because of their high rates of subsequent cardiovascular events and death .However, observational studies from routine care show that many patients do not receive high‐intensity LLT as first‐line therapy, fail to be switched to high‐intensity LLT, and have suboptimal adherence rates.The data analyzed in this study are available from the corresponding author upon reasonable request.International Classification of Diseases, Tenth Revision (ICD‐10) codes for all hospital admissions, causes of death, and dispensed prescription drugs. All diagnoses were coded according to the ICD‐10. The regional ethical review board in Stockholm approved the study (EPN 2017/793–31) and waived the need for informed consent, because data made available to researchers were deidentified.We used data from the Stockholm Creatinine Measurements database, a health care use database of residents in Stockholm, Sweden.We selected all adult patients (aged ≥18 years) admitted for a first or recurrent CHD event, including hospitalization for acute myocardial infarction or coronary revascularization (coronary artery bypass graft surgery or percutaneous coronary intervention) see Data  and who Anatomical Therapeutic Chemical [ATC] code C10AA) and ezetimibe (ATC code C10AX09) before and after the CHD event , which are conditional on surviving and being on treatment. In diseases with high mortality risks, this can lead to underestimation of effects and immortal time bias. To minimize this, we considered each LLT dispensation as a time‐dependent exposure and created a rolling assessment,Previous research has examined the drawbacks of dichotomizing PDC into a binary measure.Study covariates were assessed at baseline as well as at each new LLT dispensation, and included age, sex, laboratory measurements , medical history, ongoing medications, recent health care use, days from CHD discharge to the first LLT dispensation, and calendar year. Detailed definitions for these covariates are shown in Table The primary outcome was the occurrence of MACE, a composite of nonfatal myocardial infarction, nonfatal ischemic stroke, and all‐cause death Table . The maiDemographic and clinical characteristics of the patients are presented as number and percentage for categorical variables and mean and SD, median and interquartile range, and range for continuous variables.2), history of previous CHD, baseline LDL‐C of ≥3.5 mmol/L, and prevalent LLT use to estimate multiplicative interactions by the likelihood ratio test.We used multivariable Cox proportional hazard regression with time‐varying covariates to evaluate the associations between 1‐year treatment intensity, 1‐year adherence, and their combined product  with the risk of study outcomes. Each continuous measure was divided by 10 so that each 1‐unit change in the β estimate was attributed to a 10% absolute change in continuous measures. We evaluated these exposures as continuous and categorical variables in 3 separate models, presenting adjusted hazard ratios (HRs) with 95% CIs. We evaluated the consistency of our findings through prespecified subgroup analyses, which included sex, age (<65 and ≥65 years), CHD type , diabetes, estimated glomerular filtration rate categories  for the attainment of LDL‐C goals over time. We formally tested for continuous‐by‐continuous interaction between continuous measures of adherence and treatment intensity to illustrate the relationship between the 2 measures and the probability of LDL‐C goal attainment. Data were analyzed using R version 4.0.5 .The assumption underlying the exposure intensity measure is that patients who discontinue treatment receive 0 intensity in that year, and thus the intensity measure is directly tied to the adherence measure. We performed a sensitivity analysis in which patients were censored when discontinuing treatment to analyze only patients on treatment.2 (19%). Nearly all patients in the study were treated with antiplatelet agents (99%) and a β‐blocker (91%).Figure The patients were followed for a total of 72 839 patient‐years, with a mean of 2.6±1.1 years, ranging from 1 to 7 years. During that period, 279 885 LLT dispensations were recorded Figure . The medA total of 3046 patients (15%) experienced MACE, with a mean event rate of 38 per 1000 person‐years Tables  and S7. Each 10% increment in adherence was associated with a 6% reduction in risk of MACE . This was attributed to risk reductions in all individual MACE components Table . In addiSimilarly, every 10% increase in treatment intensity was associated with an 8% reduction in the risk of MACE . As a categorical exposure, and compared with high‐intensity LLT, patients on low‐moderate–intensity treatment had a similar MACE risk , whereas patients who discontinued LLT were at a higher MACE risk  Table . ResultsWhen modeling the combined measure of adherence‐adjusted intensity, each 10% increase was associated with a lower risk of MACE by 9% , and this was also observed for secondary outcomes Table . AdherenResults were consistent across subgroups evaluated Figure , and theEach 10% increase in adherence , intensity , or the product term of adherence and intensity  was associated with higher odds LDL‐C goal attainment , the Swedish Heart and Lung Foundation (number 20190587), and the Westman Foundation.J.J.C. received institutional funding from AstraZeneca, Astellas, Amgen, and ViforPharma outside this study. C.M.C. received consultation, advisory board membership, honoraria, or research funding from the Ontario Ministry of Health, Sanofi, Pfizer, Leo Pharma, Astellas, Janssen, Amgen, Boehringer‐Ingelheim, and Baxter, and through LiV Academy and AstraZeneca. T.J. received institutional funding from Novartis outside this study. The remaining authors have no disclosures to report.Data S1Tables S1–S11Figures S1–S5Click here for additional data file.


Patients with gastroesophageal reflux disease (GERD) frequently also have functional dyspepsia (FD) symptoms, which impair their quality of life. However, the magnitude and characteristics of the effects of each symptom on daily life have been unclarified. Using multiple regression analysis, we aimed to clarify these questions.We enrolled 290 patients from 29 institutions across Japan, in this prospective, observational study. Patients responded to three questionnaires  before and after 4 weeks of proton pump inhibitor treatment. Pearson correlation and multiple regression analyses were conducted between symptoms such as typical GERD, epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS) of FD, and aspects of daily life, namely, level of satisfaction with the daily life of GERD‐TEST, anxiety and depression score of HADS, and physical and mental component summary of SF‐8.Pearson correlation analysis showed a significant correlation in all combinations between GERD/FD‐EPS/FD‐PDS symptoms and the nine aspects of daily life. However, multiple regression analysis results deviated from these results, with the most significant effects seen in the PDS‐symptom subscale (SS) on the five aspects of daily life, that is, dissatisfaction with eating, daily life‐SS, anxiety, depression, and mental component summary (MCS) whereas the significant effects in GERD‐SS on five aspects of daily life, that is, dissatisfaction for eating, anxiety, depression, physical component summary, and MCS, disappeared.Dealing with co‐existing FD symptoms without overlooking them may be important in the management of GERD. Patients with gastroesophageal reflux disease (GERD) frequently also have co‐existing symptoms of functional dyspepsia (FD). The authors aimed to investigate the effects of each symptom of typical GERD, co‐existing FD epigastric pain syndrome (EPS), and postprandial distress syndrome (PDS) on patients’ level of satisfaction in daily life, the presence of psychiatric disorders, and quality of life using multiple regression analysis. Of the three symptoms, FD‐PDS symptom had the most prominent effect on the various aspects of daily life, whereas the impact of GERD symptom on daily life was rather modest. Gastroesophageal reflux disease (GERD) is common and frequently encountered in daily clinical practice. GERD symptoms are known to affect various aspects of daily life,This was a multicenter, prospective, observational study conducted across 29 Japanese institutions, where one or more investigators per institution was a member of the Society for GERD, a Japanese collaborative research group consisting of GERD specialists. The study was conducted in accordance with the Declaration of Helsinki , after approval by either the relevant institution's ethics committee or the central ethics committee of Nishi Clinic, Osaka, Japan. The study was registered with the University Hospital Medical Information Network Center Clinical Trials Registry in Japan (reference number UMIN000006614). The data collection period of this study was from April 2011 to March 2012. Written informed consent was obtained from all enrolled patients.Outpatients with symptomatic GERD who received proton pump inhibitor (PPI) treatment during routine clinical care were recruited. After endoscopic examination, patients were treated with a PPI at a dosage approved in Japan before the start of this study (April 2011), that is, omeprazole 20 mg, lansoprazole 30 mg, or rabeprazole 10 or 20 mg once daily. Inclusion criteria were: (i) moderate or severe heartburn or acid regurgitation at least once a week or mild heartburn or acid regurgitation at least twice a week during the 2 weeks prior to the start of the study ;Exclusion criteria were (i) comorbidity or history of disease that could potentially affect the study results ; (ii) concurrent symptoms of concern such as vomiting, peptic ulcer—except those in the scarred stage—and mental disorders; severe hepatic, renal, or cardiac diseases; uncontrolled metabolic diseases; neurological diseases; collagen diseases; or other diseases; (iii) confirmed or suspected malignancy; (iv) history of gastrointestinal tract resection or vagotomy; (v) history of hypersensitivity to PPIs or their excipients; (vi) Helicobacter pylori eradication within 6 months before enrollment; (vii) pregnancy, possible pregnancy, or breastfeeding; (viii) ingestion of PPI or histamine type2 (H2)‐receptor antagonist within 1 week of enrollment; and (ix) patients otherwise deemed to be ineligible by the attending physician. Prohibited concomitant drugs were those that might affect the study results  and drugs that might interact with the study drugs.Severity of reflux esophagitis was assessed according to the modified Los Angeles classification system.Patient characteristics were recorded using a questionnaire that included sex, age, height, weight, and lifestyle factors . The GERD‐TEST questionnaire is a patient‐reported questionnaire composed of 13 items for investigating GERD and dyspeptic symptoms, its impact on the patient's daily life, and patient's impression of the therapy. Questions (Q) 1 to 5 of the GERD‐TEST assess the severity of GERD and FD symptoms; Q6‐9 assess the impact of symptoms on daily life, including eating, sleeping, daily activity, and mood; Q10–12 evaluate the therapeutic response to the PPIs; Q13 asks about medication compliance; Q1–11 and Q13 use a Likert scale; while Q12 uses a numeric rating scale (NRS)  was defined as the mean score for heartburn (Q1) and regurgitation (Q2). The FD‐EPS‐Sx (symptom) was defined as scores for epigastric pain/burning (Q3) and the FD‐PDS‐SS was defined as the mean of scores for postprandial fullness (Q4) and early satiation (Q5). The dissatisfaction with daily life‐SS was defined as the mean of scores for dissatisfaction with eating (Q6), sleeping (Q7), daily activities (Q8), and mood (Q9).We set the followings which were measured before treatment as evaluation items in this study: GERD‐SS, FD‐EPS‐Sx, and FD‐PDS‐SS; which express the severity of each symptom of GERD and FD, dissatisfaction with eating, sleeping, daily activity, mood, and daily life‐SS; which express the patients' level of satisfaction for daily life, HADS anxiety and depression; and the SF‐8 PCS and MCS. We then investigated the correlation between the GERD‐SS, FD‐EPS‐Sx, and FD‐PDS‐SS symptoms and the various aspects of daily life  as well as the effect magnitude of these symptoms.r (Pearson correlation coefficient) and β (standardized regression coefficient) ≧0.100, R2 (coefficient of determination) ≧0.020: small; r and β ≧ 0.300, R2 ≧ 0.130: medium; and r and β ≧ 0.500, R2 ≧ 0.260: large. Data analysis was undertaken using JMP12.0.1 software . The values of P < 0.05 were considered significant.Pearson correlation analysis was performed to examine the correlation between symptoms  and daily life . In addition, we added age, sex, and BMI as explanatory variables to perform multiple regression analysis to identify the effect of individual symptoms on the above various aspects of daily life. The effect sizes of the analyses were interpreted as 2; and 178 males (61%). The severity of esophageal mucosal injury by endoscopy was as follows: non‐erosive reflux disease (NERD), 107 patients (37%); and erosive reflux disease (ERD), 183 patients (63%), with more ERD patients , Q2 (acid regurgitation), Q3 (epigastric pain/burning), Q4 , or Q5 (early satiation), results showed that there were 219 patients (75.5%) with the co‐existence of GERD symptoms (Q1 and/or Q2) and FD symptoms (Q3 and/or Q4 and/or Q5). The breakdown was as follows: co‐existence with EPS symptoms, 39 patients (13.5%); co‐existence with PDS symptoms (11.7%); and co‐existence with both symptoms, 146 patients (50.3%) (Table P < 0.001) , HADS anxiety and depression, and SF‐8 PCS and MCS (1) Table .Multiple regression analysis results greatly deviated from the Pearson correlation analysis results. For GERD symptoms, five out of nine various aspects of daily life  were no longer significantly affected. For FD‐EPS symptoms, depression was no longer significantly affected; and for FD‐PDS symptoms, PCS was no longer significantly affected. Additionally, there was a diminished magnitude of the effect of each symptom in the multiple variable aspects of daily life. Age, sex, and BMI had no significant effect on various aspects of daily life, except that PCS was significantly better in males.The items for the level of satisfaction for daily life that was significantly affected by the three symptoms, in descending order of effect: dissatisfaction for eating; FD‐PDS‐SS, and FD‐EPS‐Sx, sleeping; GERD‐SS, FD‐EPS‐Sx, and FD‐PDS‐SS, daily activity; GERD‐SS, FD‐PDS‐SS, and FD‐EPS‐Sx, mood; GERD‐SS, FD‐PDS‐SS, and FD‐EPS‐Sx, and daily life‐SS; FD‐PDS‐SS, GERD‐SS, and FD‐EPS‐Sx, respectively Table .The items for which psychiatric disorders were significantly affected by the three symptoms, in descending order of effect: anxiety; FD‐PDS‐SS, and FD‐EPS‐Sx, and depression; FD‐PDS‐SS, respectively Table .The QOL items that were significantly affected by the three symptoms, in descending order of effect: PCS; FD‐EPS‐Sx, and MCS; FD‐PDS‐SS, and FD‐EPS‐Sx, respectively Table .Of the three symptoms, FD‐PDS had the most prominent effect, with the most significant effects on five out of nine various aspects of daily life .GERD is known to be highly associated with FD,Helicobacter pylori.The prevalence of GERD in Japan has significantly increased since the latter half of the 1990 s,The co‐existence of FD has been reported to be common in GERD patients, with an incidence between 9–63.8%.The GERD‐TEST has been developed and validated by the study committee of the GERD SocietyBesides symptoms, background factors such as age, gender, and BMI may also affect various aspects of daily life. Therefore, age, sex, and BMI were also added to the three symptoms as explanatory variables for multiple regression analysis. As a result, these background factors had little effect on daily life Table .In the future, investigations of the effects of gastrointestinal symptoms on daily life should also incorporate co‐existing symptoms and examine the effects of each symptom using multiple analyses. Patients who complained of GERD symptoms reported adverse effects on various aspects of their daily life, but multiple regression analysis results showed that the co‐existing FD symptoms—rather than GERD symptoms—had a noticeable effect that could not be ignored. Particularly, FD‐PDS‐SS had a large effect on dissatisfaction with eating, psychiatric disorders such as anxiety and depression, and the degree of mental health in SF‐8.A limitation of this paper is that it only investigated the effects of typical symptoms of GERD and FD on daily life. It has been suggested that atypical symptoms of GERD and FD have a considerable effect on the daily life of patients;We showed that co‐existing FD symptoms have a significant effect on various aspects of the daily life of GERD patients. It is postulated that investigating not only the typical GERD symptoms, but also the co‐existing FD symptoms, and understanding the magnitude and characteristics of each symptom in various aspects of daily life, would be useful in estimating the burden in the patient's daily life and mental health. This could provide more appropriate treatment options in the management of GERD.


Surrogate rapid serological assay was urgently demanded for accessibly interpretation of immunity potency and duration of neutralizing antibody against SARS‐CoV‐2. The longitudinal trajectory of antibody profile with a reliable large‐scale assay was crucial to judge the protective immune status, avoid futile therapy and provide insight into the booster vaccination minimizing the risk of COVID‐19.A total of 195 volunteers were enrolled for a two‐doses procedure (0 and 28 days) of inactive vaccination, as well as ten COVID‐19 convalescents. The serum was collected at six time point and detected by chemiluminescent immunoassay with SARS‐CoV‐2 neutralizing antibody (Nab), SARS‐CoV‐2 RBD immunoglobulin G (IgG) antibody (RBD IgG) and RBD total antibody. The diagnostic results and the correlation of antibody level were evaluated among three serological  assay, as well as with an authorized cPass kit (Nab). Referred to the assay‐specific threshold, the seroconversion rate and dynamic titer of antibody were exhibited from 0 to 56 days since vaccination.p > 0.05). Both diagnostic results of neutralizing and RBD IgG antibody testing differentiated from RBD total antibody assay (p < 0.05). The coefficient of correlation (R) was above 0.90 among the levels of those three antibodies, more than 0.60 in comparison with neutralizing antibody by cPass enzyme‐linked immunoassay. The “S” varying pattern for various antibodies level was observed with time extension after vaccination. The seroconversion rate was below 11.1% in 2 weeks after the priming dose, while the value climbed to 81% in 1 week after the boosting dose. The seroconversion rate was maintained around 91%. The inactive vaccine elicited 81‐fold higher antibody levels after finished the vaccination schedule than that at the basic point. Besides, the level of neutralizing antibody induced by vaccine was found with a 0.2‐fold ratio by comparison with that in COVID‐19 convalescents.There was no difference observed with diagnostic results between neutralizing and RBD IgG antibody (The humoral immune response products including SARS‐CoV‐2 neutralizing, RBD IgG antibody and total antibody and the varying pattern of the antibody profile could be rapidly detected by CILA method. Meanwhile, the continuing and dynamic determination was attributed to evaluate the protection effect of humoral immunity against the SARS‐CoV‐2 infection. The humoral immune response products including SARS‐CoV‐2 neutralizing, RBD IgG antibody and total antibody and the varying pattern of the antibody profile could be rapidly detected by CILA method With extensive vaccination, much attention has been paid to study on the persistence of antiviral serum antibodies after vaccination and in convalescent patients. Particularly, protective Nabs present in the body preferentially binding to epitopes  in S protein of SARS‐CoV‐2, thereby blocking the invasion of the virus into the angiotensin‐converting enzyme 2 (ACE2) on the surface of human cells.22.1Participants were recruited in Shenzhen Luohu People's Hospital from December 2020 to February 2021. The vaccination group aged at 18 and 59 was confirmed by PCR detection and an epidemiology history within 14 days. The participants were included and received a schedule with two‐dose inactivated SARS‐CoV‐2 vaccine. On a voluntary basis, a 2‐month visit was performed with serological detection. Exclusion criteria was following, (1) only one vaccination was received; (2) the interval between two dose was less than 4 weeks. A total of 195 participants were enrolled, including 86 males and 109 females with an average age of 39 years. Among them, 37 cases completed a 2‐month follow‐up, 158 and 122 participants were included for serological analysis at days 14 and 28 after finished the whole vaccination procedure, respectively. Moreover, 4 males and 6 females of the convalescent patients were included with clinical diagnosis and consecutive PCR results. The study was obtained written informed consent and approved by the Ethics Committee of Shenzhen Luohu People's Hospital (2020‐LHQRMYY‐KYLL‐033).2.2−1 for 5 min, and was stored at −80°C until serological assays.Volunteers were inoculated with the inactivated SARS‐CoV‐2 vaccine provided by China National Pharmaceutical Group Co., Ltd. (Sinopharm) through intramuscular injection of the left upper arm. Two doses were given at an interval of about 28 days. In a single‐center, randomized, double‐blind trial, venous blood (3 ml) was collected with a six‐visit, including before the first dose as the baseline sample (recorded as 0 day), 14 and 28 days after first‐dose (14 and 28 days), and 7, 14, and 28 days after the second vaccination , respectively. The serum was separated by 3000 r·min2.3−1 was defined as a positive case in serological test, conversely, was a negative sample. Each SARS‐CoV‐2 specific antibody was detected as the following principle.Three SARS‐CoV‐2 specific antibodies including Nabs, RBD IgG and total RBD antibody were performed on automatic chemiluminescence immunoanalyzer (Mindray CL‐6000i). The ancillary antibody kits , calibrator and quality control substance were provided by Shenzhen Mindray Biomedical Electronics Co., Ltd. All the testing processes were strictly operated in accordance with the instructions, and the sample volume was 10 μl. As the Manufacturer's interpretation, the result above 10 AU·mlNabs detection is based on competitive CLIA. The diluted serum was firstly incubated with recombinant RBD antigen coated with magnetic beads at 37°C. SARS‐CoV‐2 Nabs present in the sample specifically bound to the recombinant RBD antigen on the surface of magnetic beads. Then, the above mixture was incubated with ALP‐ACE2 complex at 37°C, and Nabs competed with ALP‐ACE2 to bind recombinant RBD antigen. Excessive reagent was discarded, the reaction substrate AMPPD was added. The luminous value of CLIA method was inversely proportional to the content of Nabs in the sample, and its content was calculated. RBD total antibody was detected by a double antigen sandwich method on the CLIA platform. Different from Nab analysis, the detection of RBD total antibody applied an ALP‐RBD complex, accordingly, the luminous value was directly proportional to the RBD total antibody content. An indirect CLIA was used for RBD IgG antibody detection. Instead of ALP‐ACE2 complex, the mouse anti‐human IgG labeled with ALP was added and captured the specific IgG antibody fraction.Reference reagent cPass, SARS‐CoV‐2 Nabs detection reagent is based on the competitive ELISA . In strict accordance with the instructions, the serum (100 μl) was diluted with buffer at 1:9, afterward, the diluted sample was incubated with the same proportion of HRP‐RBD solution at 37°C for 30 min. Then, 100 μl of mixture was added to the capture plate (precoated with hACE2 protein) and incubated at 37°C for 15 min. Finally, the plate was washed with cleaning solution for four times, and the absorbance was recorded at 450 nm after adding TMB solution. Positive and negative quality control were prepared in parallel for inhibition rate. The cutoff with an inhibition rate ≥30% was determined as SARS‐CoV‐2 Nabs positive one.2.4p < .05 was considered to be statistically significant. Passing‐Bablock regression was used to calculate the correlation between the two methods. In view of magnitude discrepancy between ELISA and CLIA, the data was standardized processing with its cutoff value before the Spearman correlation coefficient analysis.The results were statistical analysis by SPSS 23.0, GraphPad Prism 7.0 and Python. The seroconversion rate was calculated at six‐visit point. McNemar test and paired Wilcoxon test were applied to analyze the differences between groups as well as paired samples, respectively. A 33.1p = .70 > 0.05). Whereas, the diagnostic results of RBD total antibody differed from that of Nabs and RBD IgG . In comparison with Nabs by cPass, RBD total antibody showed no difference (p = .19). On the whole, the assessment of Nabs, RBD IgG and RBD total antibody reached a good agreement with each other (Cohen's Kappa not <0.81). For antibody titer, Nabs, RBD IgG and RBD total antibody by CLIA mutually obtained an wonderful correlation, while a sighter week correlation with Nab by cPass .The seroconversion rates of three SARS‐CoV‐2 antibodies at six‐visit were summarized in Table 3.2p < .05), especially for Nab level. Compared with the baseline value of 1.272 AU/ml , Nabs levels were increased by threefold and sixfold at 14 and 28 days postvaccination, and rapidly were grown to 61.81 AU/ml (95% CI: 36.94–86.68 AU/ml) after a booster immunization. The maximum 81‐fold increase to 104.1 AU/ml (95% CI: 76.54–131.6 AU/ml) was monitored at 14 days after the second vaccination.As shown in Table 3.3n = 46) from convalescent patients were 100%. The antibodies titer changes in ten participants were plotted according to subgroups with the time of onset and recovery time, as shown in Figure The seropositive rates of RBD total antibody, RBD IgG and Nabs in all serum samples  with cPass was found a slightly better than that of total antibody (R = 0.60). Passive immunity evoked by the inactivated PiCoVacc were similar to those produced by COVID‐19 convalescent patients, RBD antibodies predominated and the key neutralization domain (CND) in RBD structure was found inducing a strong and extensive neutralization response.R = 0.689) between Nabs titer with the plaque reduction neutralization test (PRNT50) and SARS‐CoV‐2 RBD IgG titer (Snibe diagnostics) with ELISA in 218 convalescent patients. The RBD total antibody detected by a double antibody sandwich method on electrochemiluminescence platform related to the Nabs by PRNT50, with an agreement at 0.56 of Cohen' kappa.50 in development phase, the characteristics comparison indicated RBD IgG, total antibody ad Nabs on the rapid, automatic and high‐throughput CLIA may be expected to be used an alternative tool of PRNT50 for large‐screening the ability of SARS‐CoV‐2 clearance, the risk of reinfection and the passive immune response of vaccine.In the study, Nab and binding antibodies including RBD IgG and total antibody were analyzed on an automatic CLIA platform. Lacking of VNT as a standard, Nabs in cPass (ELISA method) authorized by Food and Drug Administration with an emergency schedule was applied as a reference. As expected, Nabs, RBD IgG, and RBD total antibody analysis on CLIA obtained an excellent negative and positive agreement with the reference kit by 99.2%, 98.5%, and 98.5%, respectively. For titer observation, the correlation of RBD IgG and Nabs  stimulated the body producing immune response by encoding SARS‐CoV‐2 S‐RBD protein. The Nabs titer and RBD IgG level increased rapidly at 7 days but then decreased at 21 days after booster immunization. Besides, in comparison with convalescent patients, the Nabs titer through passive immunity was 0.7–3.5 times, and RBD IgG was 6.5–30 times.Plasma therapy with COVID‐19 convalescent patients worked through a high concentration of specific antibody in plasma to neutralize SARS‐CoV‐2, activate complements, and produce an effective immune response. Hence, the antibodies at adequate level laid a foundation for plague prevention and treatment. Equally, the vaccine induced to produce abundant Nabs, which effectively reduced the infection and severe rate. Herein, an automatic CLIA platform were verified to quickly evaluate Nabs, RBD IgG, and total antibody in humoral immune response for vaccine donors and convalescent patients. Moreover, the longitudinal profile of neutralizing and binding antibodies was monitored, and laid a foundation for illustrating the production rules. The study may be helpful to understand how immunity responses against SARS‐CoV‐2 and to offer deep insight into vaccination and therapy for COVID‐19.Xiaowen Dou: conceptualization, writing‐original draft, software. Enyun Wang: methodology, validation. Ruiwei Jiang: Fromal analysis, Validation. Min Li: software, data curation. Dan Xiong: investigation, resources. Baoqing Sun: writing‐review and editing. Xiuming Zhang: supervision, project administration.The authors declare no conflicts of interest.


Since the start of the coronavirus disease 2019 (COVID‐19) pandemic, new variants of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) have emerged, accelerating the spread of the virus. Omicron was defined by the World Health Organization in November 2021 as the fifth “variant of concern” after Alpha, Beta, Gamma, and Delta. In recent months, Omicron has become the main epidemic strain. Studies have shown that Omicron carries more mutations than Alpha, Beta, Gamma, Delta, and wild‐type, facilitating immune escape and accelerating its transmission. This review focuses on the Omicron variant's origin, transmission, main biological features, subvariants, mutations, immune escape, vaccination, and detection methods. We also discuss the appropriate preventive and therapeutic measures that should be taken to address the new challenges posed by the Omicron variant. This review is valuable to guide the surveillance, prevention, and development of vaccines and other therapies for Omicron variants. It is desirable to develop a more efficient vaccine against the Omicron variant and take more effective measures to constrain the spread of the epidemic and promote public health. In this article, we described the discovery of the Omicron variant, biological characteristics of the Omicron variant, immune escape, mutations, patients’ clinical features, vaccination, detection methods, treatment with drugs and neutralizing antibodies, and protective measures taken. It is desirable to develop a more efficient vaccine against the Omicron variant and take more effective measures to contain the spread of the epidemic and promote public health. Under the wall of vaccines, antibodies, and drugs, variants continue to arise, including five variants of concern (VOCs)  with increased transmissibility, virulence, or reduced diagnostic, therapeutic, and vaccine potency, which pose a potential threat to public health.Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has swept the world in various forms, with different mutations. As of July 22, 2022, there were 565,207,160 cases of coronavirus disease 2019 (COVID‐19) and 6,373,739 deaths . Omicron was a newly emerged VOC, which possessed seven subvariants, including BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.4, and BA.5. Currently, Omicron BA.2 is the main variant causing SARS‐CoV‐2 infections worldwide. However, the SARS‐CoV‐2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants are phylogenetically independent of the BA.2 evolutionary branch. The Omicron BA.4 and BA.5 subvariants are currently at low endemic levels globally. The rapid spread of new subvariants of Omicron has led to a rapid increase in prevalence in the USA and South Africa, implying its potential for further global pandemics. The new subvariants of Omicron have unique mutational sites, which facilitate accelerating virus transmission. BA.4 and BA.5 were found in South Africa in December 2021 and January 2022, respectively.The emergence of Omicron, especially the new subvariants, may reduce the effectiveness of current drugs and COVID‐19 vaccines. Omicron variants have attracted much attention worldwide and pose a serious threat to public health. This review aims to introduce Omicron variants in a more comprehensive, detailed, and timely manner, the characteristics of Omicron, mutation sites of known subvariants, and new subvariants. Meanwhile, the protective efficacy of existing drugs, antibodies, and COVID‐19 vaccines to Omicron will be summarized and may help to block the Omicron transmission and provide relevant theories and countermeasures for COVID‐19 therapy and ending epidemics.22.1https://www.who.int/news/item/26‐11‐2021‐classification‐of‐omicron‐(b.1.1.529)‐sars‐cov‐2‐variant‐of‐concern ). Until July 16, 2022, Omicron spreads to at least 185 countries all over the world  was initially sequenced in South Africa. It was first reported by the World Health Organization (WHO) on the November 24, 2021, and then designated as a VOC by WHO on November 26, 2021 . According to statistics from Denmark as of February, Omicron BA.2 was responsible for 88% of all SARS‐CoV‐2 cases, and BA.2 may even evade BA.1‐induced natural immunity.https://www.cdc.gov/coronavirus/2019‐ncov/index.html ). In addition, compared with BA.1, BA.2 could cause more severe diseases in hamsters.It should be noted that BA.2 tended to outperform BA.1. Israeli news indicated that BA.2 was approximately 30% more infectious than BA.1  showed that people infected with Omicron are 40–45% less likely to require hospitalization than Delta. A survey by a hospital in South Africa showed that only one‐third of patients infected with Omicron developed pneumonia symptoms, 72% of pneumonia patients had mild to moderate disease, and 45% of patients needed oxygen supplementation. Clinical symptoms caused by Omicron appear to be diminishing compared with the Alpha.https://www.research.ed.ac.uk/en/publications/severity‐of‐omicron‐variant‐of‐concern‐and‐vaccine‐effectiveness ). In addition, children may be more susceptible to Omicron than variants preceding it. In the United States, children account for approximately 5% of all COVID‐19 hospitalization cases caused by Omicron, which is fourfold higher than the previous wave ,https://www.who.int/publications/m/item/enhancing‐readiness‐for‐omicron‐(b.1.1.529)‐technical‐brief‐and‐priority‐actions‐for‐member‐states ). It is worth nothing that in areas ravaged by Omicron, most people have a history of SARS‐CoV‐2 infection and have received at least one dose of the COVID‐19 vaccine; therefore, milder disease severity does not indicate reduced virulence of Omicron, considering previous immunization could protect against serious diseases.Preliminary data from South Africahttps://www.the‐scientist.com/news‐opinion/omicron‐propagates‐70‐times‐faster‐than‐delta‐in‐bronchi‐study‐69540 ). Differences in the pattern of Omicron infection generated by abundant mutations were strongly associated with changes in the transmission rate and clinical severity. Omicron can enter cells effectively through the endosomal pathway independent of TMPRSS2 and is weak in inducing syncytial formation.The clinical symptoms of Omicron vary owing to plentiful mutations. To study Omicron replication, Meng et al.2.4Omicron carried a large number of mutations. The essential amino acid substitutions in spike protein are highlighted in red: A67V, del69‐70, T95I, del142‐144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. Fifteen of these were located in the receptor‐binding domain (RBD): G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H  has been shown to enhance binding affinity.https://www.cms.gov/monoclonal ). Sotrovimab appears to be minimally affected by mutations in Omicron with a twofold to threefold neutralizing ability reduction, while the rest of the therapies are remarkably affected.Mutations in RBD are also responsible for viral evasion by clinical monoclonal antibodies. Of the five currently United States Food and Drug Administration (US FDA)‐approved emergency use authorization monoclonal antibody therapies , REGEN‐COV and the combination of Bamlanivimab and Etesevimab were eliminated as a treatment or postexposure prophylaxis for COVID‐19 due to the advent of Omicron . BA.2 contains no 69/70 deletions and therefore cannot be detected by SGTF.The numerous mutations resulting in altered local conformation, charge, and hydrophobic microenvironment of Omicron spike render them unrecognized by most NTD and RBD antibodies, leading to viral immune escape.3Due to the large number of variants in Omicron, appropriate detection methods are urgently needed. The main detection methods for Omicron are standard polymerase chain reaction (PCR) assays, reverse transcription‐PCR (RT‐PCR) assays, multiplex qRT‐PCR assays,Kozlov4Several studies have shown that the Omicron variant has significantly more immune evasion properties than its predecessor.4.1As expected, convalescent sera from COVID‐19 patients were resistant to various variants, with Omicron conferring the strongest immune escape.Several independent teams have also successively demonstrated that only 0–47% of the convalescent serum samples from WT infected patients have neutralizing activity against Omicron.n = 18).Whether SARS‐CoV‐2 variants can cause cross‐protection after infection is also an issue of widespread concern. Studies have found that the neutralizing activity of serum against Omicron is still limited after infection with most variants. The proportion of Omicron neutralizing activity from B.1.1.7 (Alpha) infected patients ranges from 0 to 40%,Sera from patients infected with the B.1.617.2 (Delta) variant appear to provide broader cross‐neutralization of Omicron; 0–85.7% of samples had detectable Omicron neutralization titers above the threshold.In addition, and somewhat unexpected, the cross‐neutralizing response induced by infection with the Omicron variants appeared to be limited. Ott et al.4.2It is generally accepted that mRNA vaccination produces higher antibody‐neutralizing titers that peak around 28 days and decline significantly after 6 months, but also generate remarkably lower or even no neutralizing activity against Omicron.Pfizer and BioNTech reported that the neutralization titer against the Omicron variant in the serum of individuals receiving two doses of their COVID‐19 vaccine decreased by more than 25 times.Studies have shown that the serum from mRNA‐1273 vaccine recipients appears to have slightly better neutralizing activity than that from Pfizer/BioNTech BNT162b2 vaccine recipients among healthcare workers (HCWs).The neutralization activity of serum to Omicron in patients vaccinated with two doses of BNT162b2 vaccine decreased about 5.9‐41.4 times compared with that of WT.In conclusion, there seems to be no significant difference in the neutralization effect against Omicron between the serum from people who received two doses of mRNA‐1273 and BNT162b2 vaccinees. In addition, WT and Omicron variant RBD binding did not differ significantly between the two doses of mRNA‐1273 or BNT162b2 vaccinated sera in the ELISA experimental group. The binding ability between Omicron RBD and the sera vaccinated with two doses of mRNA‐1273 or BNT162b2 vaccine was 2.9 times and 1.5 times lower than that of WT RBD and these sera, respectively.4.3Relevant studies have shown that the adenovirus vector vaccine ChAdOx1‐S/nCoV‐19 and Ad26.COV2.S had significantly less neutralizing activity than the mRNA vaccine at 28 and 56 days postvaccination.4.4Inactivated vaccines are currently the most widely used COVID‐19 vaccines, but they may have minor protection against Omicron infection.4.5Heterologous vaccination is uncommon in two doses vaccinated populations, and some studies showed that heterologous vaccination seem to enhance neutralizing activity against Omicron. Sera collected within one month after vaccination with heterologous ChAdOx1‐S/BNT162b2 showed higher neutralizing activity than sera collected within three months after vaccination with BNT162b2/BNT162b2.4.6Studies have shown that the revaccination of patients infected with SARS‐CoV‐2 can significantly increase the neutralizing activity against Omicron. Nine out of ten samples that received a single dose of vaccine following infection had Omicron neutralizing activity.4.7Based on current evidence, it is widely believed that a booster dose against COVID‐19 provides further protection to vaccinated individuals and that booster doses are critical regardless of the circulating variant.Clinical data have also shown that the booster dose reduces the infection risk and morbidity ratio of Omicron to a certain extent. The CDC stated that on January 21, 2022, the third dose of the mRNA vaccine prevented 82% of patients infected with Omicron from entering the emergency room or urgent care and 90% from being hospitalized.4.7.1The neutralizing antibodies against Omicron had a significant (20–30‐fold) increase a few months later with a third dose of the same vaccine boost, but only a modest (onefold to fourfold) increase against the WT.Overall, the mRNA vaccine booster substantially boosted the neutralizing activity of the recipients’ sera, and the neutralizing activity was still largely retained over time. However, the neutralizing antibody titers against Omicron were still lower than the WT. Vaccine effectiveness increased to 67.2% 2–4 weeks after the third BNT162b2 booster dose, with 45.7% remaining after 10 weeks or longer. And after the mRNA‐1273 homologous boost for 2–4 weeks, vaccine effectiveness reached 66.3%.Approximately 1 month after mRNA booster vaccination, the neutralizing activity against the Omicron live virus of serum was 17.6‐fold higher than that of the two doses of mRNA vaccinees’ serum. And the neutralizing activity of serum from mRNA booster vaccinees against the Omicron pseudovirus increased by 23.4‐fold compared with that against the WT from the second dose of mRNA vaccinees.The three doses of ADZ1222 vaccine increased 2.7‐fold than the second dose in the serum neutralization activity against Omicron 28 days after vaccination, although the neutralization activity against Omicron was 12.7 times lower than the Victoria.Similarly, the inactivated vaccine homologous booster provided more protection to Omicron to a certain extent, and three doses of inactivated vaccine significantly improved the neutralization effect against Omicron, with 46.7% effectiveness against Omicron at 5–9 weeks after inactivated booster vaccinationIn individuals vaccinated with the RBD‐based protein subunit vaccine ZF2001, 58.3% (seven out of 12) of sera sampled 15–60 days after the third dose could not neutralize Omicron. And the geometric mean titers  of these sera against Omicron were 9.4‐fold lower than those against D614G.4.7.2Previous studies have found that heterologous booster seem to induce a stronger, longer‐lasting immune response.4.8n = 6), two doses of BNT162b2 vaccine followed by a booster shot of CX‐024414 or ChAdOx1‐S‐BNT162B2 vaccine) were comparable to that four weeks after the second dose of vaccine.Omicron breakthrough infection after vaccination also further increased the level of neutralizing antibodies in serum, with binding antibody titers of samples from Omicron breakthrough individuals , and T cell responses induced by vaccination or infection could still recognize the Omicron variant.T‐cell immune responses elicited by infection with SARS‐CoV‐2 or vaccines are still effective against Omicron, and SARS‐CoV‐2 has not yet evolved extensive T‐cell escape mutations.4.10The neutralization activity of serum from lung cancer patients vaccinated with mRNA vaccine showed lower than that from healthy adults vaccinated with the same type of vaccine according to live virus neutralization experiments. At the same time, there was no significant difference in the antibody response to SARS‐CoV‐2 vaccination in patients receiving either PD‐1 monotherapy or a combination of chemotherapy and PD‐1 targeted therapy compared with patients receiving no cancer therapy at the time of vaccination. Similarly, the neutralizing activity against Omicron in the serum of lung cancer patients after receiving the mRNA vaccine was 79‐fold lower than that of the WT.Vaccination remains significantly associated with a substantial reduction in the risk of symptomatic COVID‐19, both during and before the Omicron wave.55.1The emergence of various variants during the COVID‐19 pandemic has created a great challenge for the development of vaccines. Antiviral drugs have more stable chemical structures compared with vaccines. The sensitivities of the Omicron to antiviral drugs are summarized in Table Remdesivir, a viral RNA‐dependent RNA polymerase (RdRp) inhibitor, has been approved by the US FDA for the emergency treatment of COVID‐19, which inhibits the replication of several RNA viruses, including coronaviruses.Corticosteroids, such as dexamethasone, were used in critically ill patients infected with the Omicron variant, especially in patients on high‐flow oxygen therapy, noninvasive ventilation, and mechanical ventilation. However, dexamethasone should not be recommended for mild to moderate COVID‐19 patients (in patients not receiving oxygen therapy).Targeted anti‐inflammatory drugs, such as IL‐6 and JAK inhibitors, can inhibit the Omicron variants. Tocilizumab effectively reduces inflammation and mortality in patients.Both WHO and related studies have indicated that JAK and IL‐6 inhibitors in combination with dexamethasone contribute to remission in patients with Omicron infection.5.25.2.1The Omicron variant contains a wide range of mutations that can escape neutralizing antibodies produced from vaccination. Nevertheless, monoclonal antibodies have the merits of high biotransformation efficiency, high specificity, high immunogenicity, and transparent mechanism of action.Compared with previous seasonal coronaviruses and influenza A, SARS‐CoV‐2 has faster evolutionary and propagation rates.Kovacech et al.RBD mutations in Omicron variants lead to escape against some monoclonal antibodies. For example, Q493R could lead to immune escape of Omicron from LY‐CoV555/CT‐P59, Q493R/S477N results in the immune escape from LY‐CoV16/ COV2‐2196/REGN10933, G339D contributes to the immune escape from S309, G446S gives rise to the immune evasion from COV2‐2130/ REGN10987, and so on.Omicron RBD has a solid binding capacity to ACE2.5.2.2Cancer patients and children are immunocompromised and susceptible to Omicron infection. Currently, vaccinations, especially the booster vaccination, offer broad protection against Omicron variants. Mohiuddin and KasaharaA related study in South Africa showed that children infected with the Omicron variant had higher hospitalization rates than those infected with other VOCs.A retrospective study conducted live virus and pseudovirus neutralization tests on the Omicron variant showed that the neutralizing effect of Omicron (B.1.1.529) variant decreased in serum from people who were vaccinated with BNT162b2 vaccine, mRNA‐1273 vaccine, Sinopharm BBIBP‐CorV vaccine, and the neutralizing activity against Omicron increased after booster vaccination.Neutralizing activity against the Omicron variant was detected in convalescent serum.All the above studies suggest that vaccination, especially booster vaccination, enhances neutralizing antibody activity against Omicron variants. Infection elicits T cell responses that cross‐recognize Omicron variantsRössler et al.6Omicron brings a more significant medical burden: In November 2021, a rapid rise of Omicron cases happened in Gauteng, South Africa.Several factors may account for the emergence of Omicron: low vaccination rates in low‐income countries, many immunocompromised individuals, and inadequate health‐related infrastructure to cope with the worsening epidemic. At the same time, the management of booster vaccines exacerbates global vaccine inequalities, so the international health agencies must take action to provide more vaccines for countries with the lowest vaccination rates and the highest susceptibility to SARS‐CoV‐2 and its variants.Coinfection: There may be simultaneous infections of Omicron and Delta, resulting in a simple combinatorial variant of double‐spiked spikes called “Delmicron.” Israel reported a woman infected with both the coronavirus and the flu named “flurona.” However, these require thorough investigation before conclusions can be drawn.Investigation of time and space: Computer models play an important role in predicting Omicron evolution and transmission trend. The Wills‐Riley model, an airborne disease risk spatiotemporal willingness model, helped to solve the spatiotemporal problem of SARS‐CoV‐2 infection risk in ventilated indoor environments.The mental impact of Omicron on COVID‐19 patients: Several studies have pointed out that the side effects of SARS‐CoV‐2 epidemics include worsening depression, anxiety, obsessive‐compulsive symptoms, and posttraumatic stress disorder symptoms; An increasing number of COVID‐19 patients exhibit extreme burnout and even suicide.7Preventive and control measures during the Omicron wave especially BA.4 and BA.5 should be more positive and scientific, measures including vaccination, increased ventilation, timely detection and isolation, wearing masks in indoor public places, washing hands, and keeping social distance from others should also be taken. Measures carried out in past influenza pandemics have provided many valuable suggestions.Public places such as schools, hospitals, restaurants, hotels, buses, subways, and movie theaters are high‐risk environments for VOC transmission. The CDC requires the PCR test results 24 h before arrival and 7‐day self‐isolation after arrival in the United States.Children and the elderly with weaker immunity are susceptible to the Omicron variant. The WHO recommends that the aged, especially those over 60 years old, should not travel during the Omicron pandemic.The authors declare that they have no conflict of interest.H. F., Y. T., L. S., and Y. Z. designed the research; A. X., B. H., and F. L. read and analyzed the papers; A. S., W. L., and X. A. participated in discussion; A. X., B. H., F. L., S. W., and H. F. wrote and revised the manuscript. All authors have read and approved the article. A. X., B. H., F. L., and S. W. contributed equally to this work.Not applicable.


Alterations in neuronal activity and cerebral hemodynamics have been reported in idiopathic generalized epilepsy (IGE) patients, possibly resulting in neurovascular decoupling; however, no neuroimaging evidence confirmed this disruption. This study aimed to investigate the possible presence of neurovascular decoupling and its clinical implications in childhood IGE using resting‐state fMRI and arterial spin labeling imaging.IGE patients and healthy participants underwent resting‐state fMRI and arterial spin labeling imaging to calculate degree centrality (DC) and cerebral blood flow (CBF), respectively. Across‐voxel CBF‐DC correlations were analyzed to evaluate the neurovascular coupling within the whole gray matter, and the regional coupling of brain region was assessed with the CBF/DC ratio.The study included 26 children with IGE and 35 sex‐ and age‐matched healthy controls (HCs). Compared with the HCs, the IGE group presented lower across‐voxel CBF‐DC correlations, higher CBF/DC ratio in the right posterior cingulate cortex/precuneus, middle frontal gyrus, and medial frontal gyrus (MFG), and lower ratio in the left inferior frontal gyrus. The increased CBF/DC ratio in the right MFG was correlated with lower performance intelligence quotient scores in the IGE group.Children with IGE present altered neurovascular coupling, associated with lower performance intelligence quotient scores. The study shed a new insight into the pathophysiology of epilepsy and provided potential imaging biomarkers of cognitive performances in children with IGE. Altered neuronal activity and cerebral vascular hemodynamics have been confirmed in idiopathic generalized epilepsy (IGE) patients, thus, the coupling between neuronal activity and cerebral blood flow may be impaired, but neuroimaging evidence on neurovascular decoupling remains scarce. Here, Jie Hu et al confirmed the impairment of neurovascular coupling (NVC) in IGE children by neuroimaging. Magnetic resonance imaging (MRI) is a reliable and noninvasive technique to study the pathological mechanisms of epilepsy in the human brain, analyzing the brain structure and function in an omnidirectional and multangular manner.Furthermore, basic research has confirmed that the blood–brain barrier is damaged in mammals with seizure.However, to the best of our knowledge, specific imaging biomarkers and neuroimaging evidence of NVC alterations in children with IGE are still lacking. Thus, the current study aimed to evaluate the neurovascular decoupling and its clinical significance in childhood IGE combining rs‐fMRI and ASL and provide a new perspective to understand the neuropathological mechanisms of this disease.22.1Ethics approval of this research was granted by the Ethic Committee of the Affiliated Hospital of Zunyi Medical University; all steps involving human subjects adhered to the Declaration of Helsinki. Parents or guardians of each participant provided written informed consent to participate in the research.The children with IGE were recruited in the outpatient department of the Pediatric Neurology Clinic of the Affiliated Hospital of Zunyi Medical University. IGE was diagnosed by two experienced specialists in pediatric neurology based on the International League Against Epilepsy (ILAE) criteria.For the control group, the inclusion criteria were (1) age 6–16 years at the time of MRI scanning; (2) no neurologic, psychiatric, or systemic illnesses; and (3) no contraindications to MRI. The exclusion criteria were (1) any unusual findings in conventional brain MR images; (2) head motion with maximum displacement >2 mm or >2° rotation visible during rs‐fMRI data preprocessing.2.2On the same day of the MRI scan, all IGE children underwent standardized neuropsychological assessments, performed by an experienced neuropsychologist. The Wechsler Intelligence Scale for Chinese Children Revised Version is an individually administered instrument for assessing the cognitive performance of children between the ages of 6 and 16 years, widely used in China.2.3After the neuropsychological assessment, the children were allowed a rest period before the imaging scans. Then, all participants underwent MRI scans using a 3.0‐T magnetic resonance scanner . A three‐dimensional brain volume sequence (3D BRAVO) was applied to obtain structural T1‐weighted images . The functional images were acquired using a gradient‐echo echo‐planar imaging sequence . The perfusion images were obtained with a pseudo‐continuous ASL sequence . Before each scan, every subject was instructed to relax, close their eyes while staying awake, and keep still.2.4http://rfmri.org/DPABI), implemented in MATLAB R2018b . The DICOM images were converted into the Nifti format. The first 10 volumes were eliminated to stabilize the signal of the images, and slice timing and realignment was performed on the remaining 200 volumes. The head motion was corrected with the Friston 24‐parameter model. Linear drift corrections were applied, and the interference of white matter, cerebrospinal fluid, and movement signals was removed using a regression. Then, the functional images were spatially normalized using a standard template in the Montreal Neurological Institute (MNI) space. The frequency range (0.01–0.1 Hz) was retained using a temporal bandpass filter. Finally, the functional images were re‐sampled into 3‐mm cubic voxels for further analysis.For all the collected rs‐fMRI data, preprocessing was performed with DPABI V5.0 2.5http://www.fil.ion.ucl.ac.uk/spm) in MATLAB was chosen for the preprocessing of these images. First, the CBF map of each participant was spatially normalized into the MNI space using the one‐step registration method, with voxel resampling to 3 × 3 × 3 mm. Then, further transformations were performed to standardize the maps, converting the CBF map of each participant into z‐scores with the following method: within the whole‐brain mask, the voxel‐wise z‐scores were calculated from each voxel's CBF value minus the mean CBF value of all voxels, divided by the standard deviation of all CBF values. Finally, the standardized CBF maps were smoothed using a 6‐mm full‐width at half‐maximum (FWHM) kernel.The CBF maps were obtained from the ASL sequence, and the SPM12 toolbox . The DC maps were then standardized into z‐scores, and smoothed with a 6‐mm FWHM kernel.Degree centrality (DC) represents the number of edge connections between a voxel and all the other voxels in the brain.2.7p values <0.05 were considered statistically significant.In each participant, across‐voxel correlation analyses were performed between DC and CBF in the whole gray matter (GM) to quantitatively evaluate the global NVC. The Pearson correlation coefficients between the CBF and DC values of all voxels within the GM mask were calculated for each participant. The coefficients from the two groups were tested for normality and variance homogeneity. Finally, two‐tailed, unpaired two‐sample t‐tests were used to compare the HC and IGE groups if the values were normally distributed, and Mann–Whitney tests otherwise. 2.8For all voxels, we calculated the CBF/DC ratio using the original values of CBF and DC, without z‐transformations, to represent the regional NVC. For each subject, the CBF/DC ratio map was then standardized into z‐scores, and smoothed using a 6‐mm FWHM kernel. We performed voxel‐wise comparisons to identify significant intergroup differences in the CBF/DC ratio. Intergroup differences in CBF and DC were also analyzed to determine the causes of the differences in the CBF/DC ratio.2.92.9.1t‐test; otherwise, the Mann–Whitney U‐test was used. Age and years of education were normally distributed and analyzed with unpaired two‐sample t‐tests. The Chi‐squared test was performed to analyze the intergroup difference in sex distribution. SPSS version 17.0  was used to perform these statistical analyses, and the significance level was set as p < 0.05.The normality of the distribution of age and years of education was assessed with the Kolmogorov–Smirnov test. Normally distributed data were analyzed with the unpaired two‐sample 2.9.2http://rfmri.org/DPABI), based on the MATLAB R2018b platform, adjusting for the covariates of age, sex, and years of education. For the resulting CBF, DC, and CBF/DC ratio maps, the Gaussian random field (GRF) method (p < 0.05) was selected to correct for multiple comparisons.The differences in CBF, DC, and CBF/DC ratio maps were analyzed using a voxel‐wise two‐sample t‐test in the DPABI software V5.0  was used to perform these statistical analyses, and the significance level was set as p < 0.05.The CBF‐DC correlation coefficients of the two groups were tested for normality using the Kolmogorov–Smirnov test; the unpaired two‐sample t‐test was used for data with normal distribution, and the Mann–Whitney 2.9.4p < 0.05).We validated our results considering four potential influencing factors: gray matter volume (GMV) changes, demographic factors , different DC correlation thresholds , and the influence of antiepileptic drugs.p < 0.05) was used to correct for multiple comparisons. We have also performed the spatial normalization for all children by using the pediatric brain templateIn addition, we replaced the DC with fractional amplitude of low frequency fluctuations (fALFF) to assess the neuronal activity. All preprocessing steps were performed using the same parameters as in the DC preprocessing. Then, the voxel‐wise CBF/fALFF ratio was calculated and compared between the two groups, controlling for the effects of sex, age, and years of education. The GRF method , some of whom were also included in our previous studies.3.2Despite subtle differences, the spatial distributions of CBF, DC, and CBF/DC ratios in the IGE group were similar to those in the HCs group Figure . A highe3.3t‐test, CBF: t = −0.208, p = 0.836; DC: t = −1.705, p = 0.093). A significant correlation was found between CBF and DC in both groups; however, the CBF‐DC coupling within the GM mask was significantly lower in the IGE group than in the HCs group  are presented in Figure 3.4p < 0.05); all results are shown in Table p < 0.05, Table p < 0.05, Table The IGE group showed significantly higher CBF/DC ratio in the right medial frontal gyrus (MFG), posterior cingulate cortex/precuneus, and middle frontal gyrus, with significantly lower CBF/DC ratio in the left inferior frontal gyrus compared to the HCs group  between the increased CBF/DC ratio in the right MFG and the performance IQ (PIQ) scores , different DC correlation thresholds , and the influence of antiepileptic drugs. The altered CBF/DC ratio was limited to the right MFG, posterior cingulate cortex/precuneus, middle frontal gyrus, and left inferior frontal gyrus Figures , S12–S14t = −0.6739, p = 0.503); however, in the regional NVC analysis, the brain regions with significantly altered CBF/fALFF ratio . The alterations of the CBF/DC ratio observed by using the pediatric brain template for spatial normalization remained consistent with our main findings, after controlling for sex, age, and years of education , Young Outstanding Scientific and technological Talent of Guizhou Province (grant No. Qiankehepingtairencai[2021]5620), and Talent Program for Future Famous Clinical Doctors of Zunyi Medical University (rc220211205).The authors declare that the study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Appendix S1:Click here for additional data file.