COMT, NAA16, CCDC22, EIF3E, AHI1, DMXL2, and CISD3) through the random forest classifier. Among these DEGs, AHI1, DMXL2, and CISD3 have never been reported to be associated with the pathogenesis of EMs. Our study indicated that these three genes might participate in the pathogenesis of EMs through oxidative stress, epithelial–mesenchymal transition (EMT) with the activation of the Notch signaling pathway, and mitochondrial homeostasis, respectively. Then, we put these seven DEGs into an artificial neural network to construct a novel diagnostic model for EMs and verified its diagnostic efficacy in two public datasets. Furthermore, these seven DEGs were included in 15 hub genes identified from the constructed protein–protein interaction (PPI) network, which confirmed the reliability of the diagnostic model. We hope the diagnostic model can provide novel sights into the understanding of the pathogenesis of EMs and contribute to the clinical diagnosis and treatment of EMs.Endometriosis (EM), an estrogen-dependent inflammatory disease with unknown etiology, affects thousands of childbearing-age couples, and its early diagnosis is still very difficult. With the rapid development of sequencing technology in recent years, the accumulation of many sequencing data makes it possible to screen important diagnostic biomarkers from some EM-related genes. In this study, we utilized public datasets in the Gene Expression Omnibus (GEO) and Array-Express database and identified seven important differentially expressed genes (DEGs) ( Endometriosis (EM) is an estrogen-dependent inflammatory disorder, which afflicts about 10%–15% of women of childbearing age . It is dDNA microarray technology is a high-throughput detection method that can be used to provide gene expression profiles and thus can help to screen disease-related genes and biomarkers . With thCOMT, NAA16, CCDC22, EIF3E, AHI1, DMXL2, and CISD3). Then, we put these seven DEGs into an artificial neural network to construct a novel diagnostic model and verified its diagnostic efficacy in two public datasets   to obtaip-value < 0.05 were set as the threshold of DEGs. The R package “pheatmap” (v1.0.12) was used to perform clustering analysis of DEGs for the heatmap. To explore the biological significance of these DEGs in the pathogenesis of EMs, GO and KEGG pathway enrichment analyses were performed through the R package “clusterProfiler” (v4.1.3)   to identhttps://string-db.org/cgi/input.pl) (The STRING (v11.5) (nput.pl)  has beennput.pl)  was emplnput.pl) .The R package “randomForest” (v4.6.14)  was usedThe GSE6364 dataset downloaded through the R package “GEOquery” was selected as the training set for the construction of the artificial neural network model. After the data normalization, the R package “neuralnet” (v1.44.2)  was used and obtain detailed information. We used the “removeBatchEffect” function in the R package “LIMMA” to adjust batch effects and then conducted principal component analysis (PCA) analysis to evaluate the performance of batch effect adjustment. PCA results  indicate+ activity, and lipid transporter activity , cellular component (CC), and molecular function (MF). The top five GO terms of genes with significantly upregulated and downregulated expression levels were visualized in activity , while tactivity . For KEGactivity , while tactivity . FurtherAHI1, DMXL2, NAA16, CCDC22, CISD3, COMT, and EIF3E) with a mean decrease of Gini index greater than 4 as important variables for subsequent analysis. Interestingly, all these DEGs were included in the 15 hub genes identified from the constructed PPI network. Among these variables, AHI1 was the most important, with the mean decrease of the Gini index being much higher than other variables  as the training set to construct the artificial neural network model. Firstly, we performed the preprocessing and normalization of this dataset. According to the output results of the neural network model , it is iectively . The larectively , the AUCectively , the AUCCOMT, NAA16, CCDC22, EIF3E, AHI1, DMXL2, and CISD3) were screened. Then, we used the GSE6364 dataset as the training set to construct the artificial neural network model and evaluated the classification efficacy of the model in E-MTAB-694 and GSE7307 datasets. The AUC values of the ROC curve were about 0.7, which had great efficiency and verified the diagnostic efficacy of the model. Furthermore, we constructed a 15-hub-gene-based PPI network and confirmed the reliability of the prediction model. Compared with the Nnet package, we found that the neuralnet package had higher accuracy of the predicted model (86.5% vs 81.1%). In total, the constructed diagnostic model could provide new insight into our understanding of the pathogenesis of EMs and identify crucial biomarkers as diagnostic and therapeutic targets of EMs.The combination of random forest and artificial neural network can be used to construct a reliable predictive model for the diagnosis of some diseases, such as polycystic ovary syndrome (PCOS)  and ulceCOMT, NAA16, CCDC22, and EIF3E have been reported to be associated with the pathogenesis of EMs. Catechol-O-methyltransferase (COMT) is highly expressed in the placental, adrenal gland, ovary, and other tissues. The degradative pathways of the catecholamine transmitters can relieve painful uterine contractions  is highly enriched in bone marrow, testis, endometrium, and other tissues. It can alter NAT 2 enzyme activity and thus contribute to the susceptibility of EMs (CCDC22), a membrane-binding protein, is highly enriched in the spleen, lymph node, and other tissues. Studies have demonstrated that there is also a relationship between CCDC22 polymorphisms and EM susceptibility (EIF3E) is highly expressed in the ovary, lymph node, endometrium, and other tissues. Its downregulation may be involved in epithelial–mesenchymal transition (EMT) in EMs, possibly through the preferential translation of snail (an inhibitor of E-cadherin)   and invoadherin) .AHI1, DMXL2, and CISD3), which have never been reported to be involved in the pathogenesis of EMs. Abelson helper integration site 1 (AHI1) is highly enriched in testis, adrenal gland, brain, prostate, endometrium, and other tissues, which has upregulated expression level in EMs. The AHI1 protein participates in reactive oxygen species (ROS) production in the form of protein complexes  encodes a protein with 12 WD domains, which has relatively low expression in endometrium tissue and downregulated expression in EMs. The DMXL2 protein is demonstrated to participate in the regulation of the Notch signaling pathway  is a member of the CDGSH domain-containing family, whose expression is upregulated in EMs. The CISD3 protein is redox active and is thought to play an important role in mitochondrial homeostasis  and non-EM  groups, which may affect the final screening results of DEGs. Secondly, the diagnostic model is only verified in two public datasets, which need more samples for verification. Thirdly, we conduct data analysis only at the mRNA level in the tissue samples of EMs, which require further validation at the mRNA and protein levels. In general, our approach has a certain clinical value, which can be beneficial for the early screening of EMs.


E2F2, E2F8, TPX2, BUB1, and CKAP2L. So it may be of great importance for further verification by cancer experts.Breast cancer is one of the most common malignant tumors in women, which seriously endangers women’s health. Great advances have been made over the last decades, however, most studies predict driver genes of breast cancer using biological experiments and/or computational methods, regardless of stage information. In this study, we propose a computational framework to predict the disease genes of breast cancer based on stage-specific gene regulatory networks. Firstly, we screen out differentially expressed genes and hypomethylated/hypermethylated genes by comparing tumor samples with corresponding normal samples. Secondly, we construct three stage-specific gene regulatory networks by integrating RNA-seq profiles and TF-target pairs, and apply WGCNA to detect modules from these networks. Subsequently, we perform network topological analysis and gene set enrichment analysis. Finally, the key genes of specific modules for each stage are screened as candidate disease genes. We obtain seven stage-specific modules, and identify 20, 12, and 22 key genes for three stages, respectively. Furthermore, 55%, 83%, and 64% of the genes are associated with breast cancer, for example  Breast cancer is one of the most common malignant tumors in women, and it is the main disease factor that causes cancer deaths in women worldwide. According to statistics , breast BRCA1 and RASSF1A methylation is higher. RUNX3 gene methylation and breast cancer, and the results showed that the hypermethylation of RUNX3 plays a significant role in the pathological stage and prognosis of breast cancer, which has great potential as a molecular marker for early diagnosis of breast cancer. PRAC2, TDRD10, TMEM132C, etc., are expected to become diagnostic and prognostic markers of breast cancer.Epigenetics is currently a promising field in cancer research. As an important part of epigenetics, DNA methylation has received increasing attention, which is the process of adding methyl groups to DNA molecules and essential for cell development. The functional epigenetic module (FEM) algorithm  has veriTP53 and PNRM1 driver genes play an important role in breast cancer through matrix tri-factorization framework with pairwise similarity constraints. There are also bioinformatics experts who study breast cancer based on biological molecular networks. In this article, we propose a computational framework to predict candidate stage-specific disease genes of breast cancer based on the stage-specific gene regulatory networks. Firstly, we screen out differentially expressed genes and hypermethylated/hypomethylated genes by comparing tumor samples and normal samples. Secondly, we construct and analyze three stage-specific gene regulatory networks by taking stage information into account. Thirdly, we identify stage-specific modules by module division. Finally, we predict candidate stage-specific disease genes.Our contributions consist of two points:(1)We integrate stage information and DNA methylation information to construct a stage-specific gene regulatory network for breast cancer, which may help doctors identify patient’s disease stage more quickly and design better treatment strategy.(2)The proposed computational framework is effective in predicting breast cancer related genes, which will help experts to explore the molecular mechanisms of breast cancer.Our computational framework for predicting candidate disease genes includes four parts: Stage-specific gene regulatory networks construction, Module division, Topological properties analysis and gene set enrichment analysis, Candidate disease genes prediction .We download breast cancer phenotype data, gene expression profile and DNA methylation data from TCGA  (The CanThe Gene Expression Omnibus (GEO)  databasep-value less than 0.05 and |log FC| less than 0.5 as the threshold. For the DNA methylation data, we define β value greater than 0.8 as hypermethylated genes and β value less than 0.2 as hypomethylated genes. Then we take the intersection of the differentially expressed genes and the hypermethylated/hypomethylated genes and obtain 1,027 genes, 1,012 genes, and 1,220 genes in stage I, stage II, and stage III, respectively. Then we compare the relationship between the DNA methylation profile and gene expression profile, and find that the higher the gene methylation level, the lower the gene expression. And the results are shown in For the gene expression profile, we use Limma  in the RGene Regulatory Network database (GRNdb)  is a genWe use WGCNA  to dividHub genes are important for biological processes. We identify and compare hub genes for each gene regulatory network. We perform topological analysis of stage-specific gene regulatory networks using Cytoscape , includiWe filter out candidate disease genes from the above modules and network topological information. Then, we checked them by known disease-related genes from OMIM, COSMIC, and DAVID. Online Mendelian Inheritance in Man (OMIM)  mainly cWe filter out the TF-target gene pairs whose target genes are not differentially expressed genes and hypermethylated/hypomethylated genes, and use the PCC cut-off 0.5 to construct stage-specific gene regulatory networks. There are 1,129, 1,066, and 1,339 nodes and 4,429, 4,879, and 6,461 edges, respectively.We use WGCNA to divide three gene regulatory networks into modules and the results are shown in We identify differentially expressed genes that only exist in one stage as the stage-specific genes and obtain 92 genes, 60 genes, and 187 genes in stage I, stage II, and stage III, respectively. Then we count the distribution of these genes in each module, as shown in We perform network topological analysis for seven specific modules using Cytoscape. For the degree distribution, the degrees of S1_turquoise module, S2_turquoise module, and S3_turquoise module are mainly distributed between 100 and 400, and the degrees of S1_brown module, S1_blue module, S3_brown module, and S3_green module are mainly distributed between 50 and 100, respectively. And the degree distribution of each module conforms to the power law distribution. The betweenness centrality of most nodes in each module is at a high level. The closeness centrality of most nodes in each module ranges from 0.5 to 0.9. These values indicate that the network corresponding to each module is a dense graph, so the hub genes screened by these three parameters are all core genes.p-value cut-off 0.01. The joint enrichment results are shown in We use Metascape to perform the joint enrichment analysis on the genes in the seven specific modules, and set p-value cut-off 0.05 as the core genes of each module. Then, we sort the degree distribution, betweenness centrality and closeness centrality of each gene in the seven modules, and select the top 5% as the core gene of each module. The intersection of core genes selected by these two methods are considered as candidate disease genes.We predict disease genes based on correlation matrix and network topological properties. Firstly, we calculate the correlation matrix of genes at each specific module, and select genes with correlation cut-off 0.8 and E2F2, E2F8, TPX2, etc., 12 genes in stage II, such as KPNA2, CKAP2L, CBX3, etc., and 22 genes in stage III, such as RAD21, FBXO5, CCNE2, etc. A complete gene list of each stage is shown in E2F2, CKAP2L and CBX3 are genes shared by three stages. For the remaining candidate genes at different stages, we compare their gene expression data and find that they are indeed different at different stages. And the results are shown in the We obtain 20 candidate disease genes in stage I, such as BUB1 is mitotic checkpoint serine, E2F2 is a transcription activator, NEK2 is a serine/threonine-protein kinase, TPX2 is the target protein for Xklp2, TTK is essential for spindle establishment and centrosome replication, PCNA is the proliferating cell nuclear antigen, and TOP2A is DNA topoisomerase 2-alpha. Most of these genes are related to cell proliferation and transcription.In order to determine whether the selected candidate disease genes are effective in the diagnosis and treatment of breast cancer, we use OMIM, COSMIC, and DAVID to verify the candidate genes, and obtain seven genes related to breast cancer. STIL is an important prognostic and predictive biomarker for triple-negative breast cancer and HER2-positive breast cancer. At present, there have been studies on pathological assessment of breast cancer based on STIL, which is a key step for molecular markers to move toward clinical treatment. Based on the study of differentially expressed hub genes, CCNE2, H2AFZ, TOP2A is closely related to the diagnosis and poor prognosis of breast cancer. CSE1L has a certain relationship with the distant metastasis of breast cancer and may be a valuable prognostic tool. FBXO5 and TPX2 are related to the poor prognosis of breast cancer. CBX family proteins have epigenetic regulatory functions, among which the high expression of CBX3 is related to the worsening of recurrence-free survival rate of breast cancer patients. E2Fs are transcription factors that affect cell proliferation, differentiation and apoptosis, and the high expression of E2F8 is also related to the deterioration of patients’ recurrence-free survival rate, and can be used as a potential target for individualized treatment of breast cancer patients. KDM5B is up-regulated in breast cancer and many other cancers and its expression is positively correlated with breast cancer metastasis. KPNA2 and SLC39A1 in breast cancer tissues is significantly up-regulated, which can regulate the development of breast cancer and provide new targets for breast cancer treatment. NEK2 is a kind of serine, which plays an important role in mitosis. NEK2 is a target for breast cancer. RAD21 can enhance the anti-tumor activity of chemotherapeutics by inducing DNA damage and is a new target for cancer drugs. Based on survival analysis and mutation analysis, CKAP2L and CASC5 is closely related to the poor prognosis of breast cancer patients. These verified genes are shown in We search the rest candidate disease genes related to the genes in PubMed, and verify whether the genes are related to breast cancer. E2F2, E2F8, TPX2, BUB1, CKAP2L, etc. The results show the effectiveness of our computational framework for predicting disease genes.In summary, we detect 20, 12, and 22 candidate disease genes for three stages, respectively. Through PubMed search, 11, 10, and 14 genes are verified, respectively. That is 55%, 83%, and 64% of the candidate disease genes are proved to be related to the diagnosis and treatment of breast cancer, respectively, such as H2AFZ, NPM1, MAF, NR3C1, PTGER3, TCF4, IRF1, RARB, CHD2, and SMAD4. Except PTGER3 and CHD2, other genes have been verified. This means that our method is effective, and it may help experts explore breast cancer related genes.We also use GSE15852 gene expression profile and GSE69914 DNA methylation profile to verify the validity of the proposed computational framework. Firstly, we screen out 79 differentially expressed genes and hypermethylated/hypomethylated genes. Secondly, we combine with the TF-target gene pairs and construct a gene regulatory network with 195 nodes and 313 edges. Thirdly, we divide the gene regulatory network into four modules: 76 genes in turquoise module, 68 genes in blue module, 18 genes in gray module, and 33 genes in brown module, respectively. In particular, the gray module contains genes that are not classified into any module and discarded. Finally, we screen the candidate disease genes of each module based on correlation matrix and network topological properties, and obtain four genes in turquoise module, four genes in blue module, and two genes in brown module, respectively. In detail, these genes are At present, the proposed computational framework has only been tested on breast cancer, and satisfactory results have been obtained. In the future, we will try to apply this framework to other types of diseases for discovering more disease-related genes.We propose a computational framework to predict candidate stage-specific disease genes for breast cancer based on stage-specific gene regulatory networks. And we conduct experiments using two breast cancer data sets and find that most predicted genes are related to breast cancer, which shows that our method is effective. We also predict some candidate disease genes that need to be further verified. Nevertheless, our research has some limitations. Our proposed computational framework is based on the public TCGA and GEO datasets, and the noise affects the analysis results. Another limitation is that we should integrate more omics data so that more disease genes may be predicted more accurately.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the Local Legislation and Institutional Requirements. Written informed consent for participation was not required for this study in accordance with the National Legislation and the Institutional Requirements.JH and LF conceived and developed the computational framework for predicting disease genes and wrote the manuscript. GQ provided important feedback in the framework process and edited the manuscript. All authors have made significant contributions to the completion and writing of the manuscript and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycotoxins in feeds can cause detrimental effects on the growth performance and health of pigs. One of the methods used to overcome the negative effects of mycotoxins in animal feeds is to add toxin sequestering agents to feed. The present work was conducted to determine the efficacy of mycotoxin sequestering agents using an in vitro method. All mycotoxin sequestering agents effectively bound to aflatoxin B1; only activated charcoal effectively sequestered deoxynivalenol; and a bentonite product, an aluminosilicate product, and activated charcoal effectively sequestered zearalenone.p < 0.05) than that of other products. The ZEA sequestering rate of bentonite F (97.0%), aluminosilicate A (99.6%), and activated charcoal (100.0%) was the greatest (p < 0.05) among the tested mycotoxin sequestering agents. Overall, most mycotoxin sequestering agents had the ability to bind to AFB1, but most products, except activated charcoal, failed to sequester DON and ZEA.The objective of this study was to determine the efficacy of mycotoxin sequestering agents for aflatoxin B1 (AFB1), deoxynivalenol (DON), and zearalenone (ZEA) using an in vitro method. The twelve toxin sequestering agents tested were seven bentonite products , two aluminosilicate products , a heulandite product, an activated charcoal product, and a yeast cell wall product. A two-step in vitro procedure was employed to mimic the conditions of temperature, pH, and digestive enzymes in the stomach and small intestine of pigs. All mycotoxin sequestering agents tested were able to bind to AFB1 with a high efficacy (>92%). The DON sequestering rate of activated charcoal (99.1%) was greater ( Aspergillus fungi, and DON and ZEA are generated by Fusarium fungi. Mycotoxins in feedstuffs have been reported to cause harmful effects on the immune system, ileal amino acid digestibility, feed intake, and body weight gain in pigs [Mycotoxins in feeds can have detrimental effects on the growth performance and health of pigs ,2,3. Afl in pigs ,2,4, ind in pigs ,6,7.One of the methods used to overcome the negative effects of mycotoxins in animal feeds is to add toxin sequestering agents to feeds . Adding Twelve toxin sequestering agents were tested: seven bentonite products , two aluminosilicate products , a heulandite product, an activated charcoal product, and a yeast cell wall product. Bentonite A, B, and E consisted mainly of calcium montmorillonite, but bentonite C, D, and G consisted mainly of sodium montmorillonite. Bentonite D additionally contained phytogenic substances, enzymes, and microorganisms. Bentonite F contained both calcium and sodium ions with an organic compound. Bentonite G additionally contained sepiolite. Aluminosilicate A consisted of a hydrated sodium silicate and calcium silicate with an organic compound. Aluminosilicate B included an activated sodium and calcium aluminosilicate. The heulandite product, a stable crystal structure with negatively charged ions, was composed of heat-treated clinoptilolite. The activated charcoal product was produced from a coconut shell. The yeast cell wall was composed of organic polysaccharides, including hydrolyzed yeast cell walls and mannan-oligosaccharides.Standard solutions of AFB1 (2 μg/mL), DON (100 μg/mL), and ZEA (100 μg/mL) in acetonitrile  were diluted to 10, 1000, and 250 ng/mL, respectively, using distilled water. The quantification ranges of the enzyme-linked immunosorbent assay (ELISA) kit used for the quantitative analysis of toxins were from 2 to 50 ng/mL for AFB1, 250 to 5000 ng/mL for DON, and 25 to 1000 ng/mL for ZEA.g for 10 min and the supernatant was collected to analyze residual unbound AFB1, DON, and ZEA. The ELISA test kits were used to determine the residual AFB1 , DON , and ZEA  concentrations. The in vitro procedures were performed in triplicate for each sequestering agent.The in vitro procedure used in the present work was based on methods mimicking the gastrointestinal tract of pigs ,21 with The sequestering rates of AFB1, DON, and ZEA by mycotoxin sequestering agents were calculated using the following equation:p-value less than 0.05.Statistical analyses were performed using the MIXED procedure of SAS . The statistical model included the mycotoxin sequestering agent as a fixed variable and the replication as a random variable. Least squares means were separated using the PDIFF option of the MIXED procedure with Tukey’s adjustment. Statistical significance was determined at a p < 0.05) than those by bentonite D and yeast cell wall  than that of other products  was greater (products . All thep < 0.05) among the tested mycotoxin sequestering agents (The sequestering rates of ZEA by bentonite F, aluminosilicate A, and activated charcoal were the greatest (g agents . HoweverFusarium mycotoxins, are often co-contaminated in animal feeds [Mycotoxins such as AFB1, DON, and ZEA are often present in feed ingredients during the growth and drying of plants in the field or during long-term storage post-harvest ,23. Mycoal feeds . In a pral feeds , five tyThe sequestering rates of AFB1 by all mycotoxin sequestering products tested in the present work agree with previous studies ,15. Clay2/g to 3500 m2/g, depending on the structure materials [The relatively low sequestering rates of DON by bentonite products are in agreement with previous reports ,16,18, iaterials . The seqaterials , which iaterials ,37. Howeaterials  had a leAmong the bentonite products, only bentonite F effectively sequestered ZEA in this work. This product has been claimed to have a high affinity to ZEA once hydrated to a polar molecule. As one of the major components of bentonite, montmorillonite in a chemically modified form has been reported to bind ZEA . HoweverIn conclusion, most mycotoxin sequestering agents used in the present work were able to bind aflatoxin B1; however, their effects on deoxynivalenol and zearalenone varied based on the in vitro procedure used to mimic the gastrointestinal tract of pigs. Only activated charcoal effectively sequestered deoxynivalenol, and a bentonite product, an aluminosilicate product, and activated charcoal effectively sequestered zearalenone.


Background: Rifaximin is commonly used for hepatic encephalopathy (HE). However, the effects of long-term treatment for Japanese people are limited. Therefore, this study aimed to investigate the effects and safety of long-term treatment with rifaximin on HE. Methods: A total of 215 patients with cirrhosis administered with rifaximin developed overt or covert HE, which was diagnosed by an attending physician for >12 months. Laboratory data were extracted at pretreatment and 3, 6, and 12 months after rifaximin administration. The long-term effect of rifaximin was evaluated, and the incidence of overt HE during 12 months and adverse events was extracted. Results: Ammonia levels were significantly improved after 3 months of rifaximin administration and were continued until 12 months. There were no serious adverse events after rifaximin administration. The number of overt HE incidents was 9, 14, and 27 patients within 3, 6, and 12 months, respectively. Liver enzymes, renal function, and electrolytes did not change after rifaximin administration. Prothrombin activity is a significant risk factor for the occurrence of overt HE. The serum albumin, prothrombin activity, and albumin–bilirubin (ALBI) scores were statistically improved after 3 and 6 months of rifaximin administration. Moreover, the same results were obtained in patients with Child–Pugh C. Conclusions: The long-term rifaximin treatment was effective and safe for patients with HE, including Child–Pugh C. Hepatic encephalopathy (HE) is a common complication of patients with liver cirrhosis, causing neuropsychological and neuromuscular dysfunctions in various degrees. HE is closely associated with a poor prognosis and reduced health-related quality of life ,2. HE isThe prognosis of liver cirrhosis reportedly worsens when overt HE occurs, with a mortality rate within 1 year of 64% and within 5 years of 85%. Covert HE occurs in 80% of patients with liver cirrhosis, and covert HE, as well as overt HE, can impair the prognosis and increase the risk of hospitalization [®; ASKA Pharmaceutical, Tokyo, Japan) was approved for the HE treatment in November 2016 in Japan. However, only data from clinical trials were available up to 12 weeks of administration [In the Japanese guideline, synthesized disaccharides are the first-line treatment for HE, which decreases the production and absorption of ammonia in the gut . One of stration ,14,15,16stration ,18,19, tThis was a multicenter retrospective observational cohort study of 215 consecutive patients with liver cirrhosis who were continuously administered 1200 mg t.i.d of rifaximin for >12 months between January 2017 and July 2020. Rifaximin was administered to patients with over Grade I coma. Patients undergoing hemodialysis were administered warfarin, and with a total bilirubin of >5.0 mg/dL were excluded from this study. The primary outcome was the effectiveness of long-term rifaximin treatment, and secondary outcomes were the evaluation of the safety of long-term rifaximin treatment.This study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and approved by the Ethical Review Committees of each participating institution for this study. Informed consent was obtained from all participants.3), sodium (Na), and potassium (K); and medical history of HE, spontaneous bacterial peritonitis (SBP), coexistence of hepatocellular carcinoma (HCC), stage of HCC, and coexistence of PSS were extracted. PSS was evaluated with enhanced CT or MRI, and a maximum vessel diameter of 5 mm indicated the existence of PSS. Moreover, the albumin–bilirubin (ALBI) score and model for end-stage liver disease (MELD) score were calculated. The occurrence or recurrence of HE was defined as the occurrence of HE Grade II or higher.Necessary data were retrospectively extracted from each participating institution before and 3, 6, and 12 months after administering rifaximin. Age; sex; height; body weight; body mass index; hemoglobin level (Hb); platelet count (Plt); prothrombin (PT) activity; serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (Alb), total bilirubin (T-Bil), blood urea nitrogen (BUN), creatinine (Cr), estimated glomerular filtration (eGFR), ammonia , and a p-value of <0.05 was considered statistically significant.Data are presented as mean ± standard deviation. To compare continuous variables at baseline and after rifaximin administration at 3, 6, 12 months, repeated measurement analysis of variance was used. A Kaplan–Meier curve was created with Gray’s test for evaluating cumulative HE occurrence. A Cox proportional hazard model was used to determine the factors associated with HE occurrence in univariate and multivariate analyses. Parameters with a The patient characteristics are shown in Ammonia levels were significantly decreased by administering rifaximin after 3 months, which was continued for 12 months A. Overt Hb, Plt, AST, and ALT were not affected at 3, 6, and 12 months of rifaximin treatment. BUN, Cr, and eGFR were also not affected at 3, 6, and 12 months of rifaximin treatment A–C. The No serious AEs, such as liver failure or ruptured esophagogastric varices, occurred for 12 months. A total of 12 patients experienced diarrhea, which was improved by the short-term rifaximin reduction or administration of probiotics. No patient had SBP during the rifaximin treatment.We evaluated the data limited to Child–Pugh C. There were 27 patients of Child–Pugh C. Ammonia levels were significantly decreased by administering rifaximin after 3 months and were continued for 12 months in patients of Child–Pugh C B. AST, Ap = 0.049) , presence of PSS, presence of HCC, Alb level at baseline, and PT activity at baseline as potential risk factors influencing HE occurrence. Multivariate Cox analysis revealed that only PT activity at baseline was significantly associated with HE occurrence  .In this multicenter retrospective cohort study, the efficacy and safety of long-term rifaximin use have been clarified. The serum ammonia level significantly decreased 3 months after initiating treatment as compared to the pretreatment level, and the effect was maintained until 12 months. Moreover, in patients with Child–Pugh C, the serum ammonia level also significantly decreased 3 months after treatment, and the effect was maintained until 12 months. Furthermore, the limited occurrence of overt HE was reported  at 12 months, suggesting that long-term rifaximin treatment has good efficacy and can be used continuously for a long time without discontinuation. This study showed that low PT activity is a significant predictor of HE occurrence. Although the West Haven criteria are mainly used to assess HE , they arTyrosine kinase inhibitors or immune checkpoint inhibitors are usually used for advanced HCC or HCC that is difficult or refractory to local treatment in patients with Child–Pugh A. A Phase I clinical trial was conducted on the use of Lenvatinib for treating advanced HCC, Grade 3, and HE occurred in 10% of the participants . PresumaRifaximin is used for Clostridioides difficile infections (CDI) and HE. Rifaximin was first approved in Italy in 1985 . HoweverIn our study, hepatic reserve such as Alb, PT activity, and ALBI score improved 3 months after rifaximin treatment. The ALBI score, which uses only Alb and T-Bil, is a useful tool for evaluating liver function ,35. StudIn this study, rifaximin was effective and useful for patients with cirrhosis (Child–Pugh C), suggesting that rifaximin is equally effective regardless of the hepatic reserve. No study has reported the hepatic reserve of rifaximin to date. The result of this study is important that the long-term rifaximin treatment is found effective and safe.One of the limitations of this study was that neuropsychological examination was not performed to diagnose covert HE, but ammonia levels were used, and the improvement of covert HE cannot be proven objectively. Moreover, the criteria for administering rifaximin differ by the attending physician and are not standardized. Previously reported studies of the Japanese population comprised a relatively small number of patients at a single center ; howeverIn the current study, liver function, renal function, and electrolytes were evaluated to assess the effects of rifaximin. AST, ALT, T-BIL, BUN, Cre, eGFR, Na, K, and MELD scores remained unchanged, whereas ALB, PT%, and ALBI scores statistically increased after 3 or more months as compared with the pretreatment, suggesting that the long-term rifaximin administration did not affect renal function and electrolytes and improve the hepatic reserve. In patients with Child–Pugh C, rifaximin administration did not affect renal function and electrolytes in the long term and improved the hepatic reserve at 3 months.Long-term rifaximin treatment showed sustained efficacy for patients with HE and resulted in no severe AEs. Moreover, rifaximin improved the hepatic reserve.


In January 2021, the Tokyo area was under pressure to provide more hospital beds for COVID-19 patients. Therefore, the metropolitan government decided to turn three public hospitals into COVID-19 treatment centers. These three hospitals subsequently suspended operation of some other treatment sections.The COVID-19 pandemic has added to already high levels of stress among medical professionals (5). As physician burnout is linked to their quality of life and reduced quality of care (7), this study aimed to analyze the factors contributing to burnout among physicians whose environments changed rapidly due to the COVID-19 pandemic.The unprecedented nature of the situation might have caused job stress and, consequently, burnout We conducted an online, cross-sectional survey among all full-time doctors between February 15 and March 5, 2021, at the three regional hospitals that were redesignated as COVID-19 treatment centers, Tokyo Metropolitan Hiroo Hospital, Ebara Hospital, and Toshima Hospital of the Tokyo Metropolitan Health and Hospitals Corporation. We used Google Forms as the survey platform. The third wave of COVID-19 had begun to subside, although Tokyo had the highest numbers of infections in Japan, and was under a state of emergency. These three hospitals are regional general hospitals in Tokyo and are designated training hospitals. The institutional review boards of the three hospitals approved this study . All participants provided informed consent.(8). No studies have validated the set cut-off to indicate burnout definitively (7).The primary study outcome, burnout, was measured using the Japanese Burnout Scale (JBS), a 17-item questionnaire with three subscales: emotional exhaustion (EE), depersonalization (DP), and lack of personal accomplishment (PA). Responses are coded on a scale from 1 (strongly disagree) to 5 (strongly agree). Higher scores indicate increased burnout p < 0.05, and all tests were two-tailed. We used a one-way analysis of variance to test for differences across multiple groups. Multiple regression analysis was performed using IBM SPSS Statistics 27.Quantitative data were analyzed using BellCurve for Excel . Statistical significance was Of the 313 full-time physicians at the three COVID-19 priority hospitals invited to participate in this survey, 161 (51.4%) responded. The subjects included a higher population of males . The largest percentage of subjects were in their 30s , followed by the 40s . Given the variety of medical departments, we divided the participants into four categories: Internal Medicine , Surgery , Outpatient and Laboratory , and residents. Working hours per week were divided into three categories to determine workload: within 40 hours/week , within 50 hours/week , and 51 hours or more/week  .p = 0.04). We found significant differences in all three JBS subscale scores between the groups in their 30s and those in their 60s, with physicians in their 30s expressing higher scores and hence, greater burnout (p = 0.006).The mean (± standard deviation) scores of the subject on the three JBS subscales were EE: 3.02 ± 1.03, DP: 2.55 ± 0.96, and PA: 3.55 ± 0.91.  burnout . Figure p <= 0.05, removal criterion: p >= 0.10) with the subscales of the JBS as objective variables and the items that showed significant associations. Four factors were associated with EE: meaningful work, work hours, sex, and change in workload after COVID-19. Work meaningfulness and work hours were related to DP, and the only factor associated with PA was work meaningfulness.(7). Conventionally, physicians are considered to have a low probability of experiencing burnout because of their high level of professionalism and independence (7). However, with the COVID-19 pandemic, the social situation has changed rapidly, and the situation of physicians has also changed.Freudenberger proposed the concept of burnout syndrome in a 1974 study of service workers, including nurses, teachers, and social workers (3). A survey of 1,961 Italian health care workers during the pandemic showed a tendency toward burnout among front-line residents, who were estimated to be highly burdened (4). We found some concordance with the literature on the influence of age on burnout (12). Many of the young doctors in our sample were concerned that they would not be able to develop their careers owing to the pandemic cutting into their practice in their specialty. Although there have been many studies on sex differences in burnout rates, there is no definitive conclusion (10). We speculated that female physicians might experience more EE than males because life events, such as pregnancy and childbirth, already cause them to have a shorter period for career development (13).Baptista et al. surveyed 214 primary care physicians during the COVID-19 pandemic in Portugal and found that sex, years of experience, depression, and anxiety correlated strongly with burnout level (14). Although all subscale scores in the present study were higher than neurologists in the previous study, the differences in DP and PA were particularly marked. In our study, EE, DP, and PA showed high standard partial regression coefficients for work meaningfulness. For physicians, saving patients by practicing their medical specialty leads to a feeling of accomplishment. Therefore, it is stressful for them to have only limited practice in their specialty (7). As previously suggested, an increase in burnout in physicians could be related to the lack of meaningful work in a situation where their expertise in their specialty cannot be utilized (16).In a survey conducted by Shimohata et al. on all members of the Japanese Society of Neurology in 2019, among the 1,261 physicians who responded, mean scores on the JBS subscales were 2.86 points for EE, 2.21 points for DP, and 3.17 points for PA (17). Our survey results on burnout identified women and younger doctors as the groups that felt more stressed. Our results also showed that loss of meaningfulness in their work can quickly lead to burnout among physicians, and the work environment of COVID-19 priority hospitals is a hotbed for this risk (15). Burnout not only reduces the quality of medical care (6), but can also result in the loss of medical staff due to increased staff turnover (5). Our survey is limited by the fact that it was a one-time survey. It is hoped that further research will reduce burnout risk among physicians working at COVID-19 priority hospitals.The COVID-19 pandemic is a global disaster that has left administrators no choice but to accommodate the changes in the functionality of their hospital. Meanwhile, the stress on the employed staff is immeasurable NoneYK created the research plan. SK drafted the manuscript. SK, YK and MK engaged in making the questionnaire. SK and YK collected data. All authors have revised and approved the final version.Tokyo Metropolitan Health and Hospitals Corporation Ebara Hospital approved this study (No.0283), Tokyo Metropolitan Health and Hospitals Corporation Toshima Hospital (Rin-rin-jin 2-47), and Tokyo Metropolitan Hiroo Hospital (Jin-73).


To provide a dynamic description of the oral microbial composition in mothers with and without dental caries and their children aging 12-24 months.A total of 20 pairs of mothers and their children aged 12 months were included and followed up at 18 and 24 months of age. Ten mothers with dental caries(MEG) and their children(CEG) were in the exposure group, and ten caries-free mothers(MCG) and their children(CCG)in control group. Supragingival plaque biofilm samples were collected and DNA was extracted for bacterial 16S rRNA gene sequencing.Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria accounted for more than 80% phyla of each group. A microbial community structure analysis showed that the differences between mothers and children were significant in all groups except for the MEG24 and CEG24 groups.A total of 18 pairs completed follow-ups. At a 3% divergence level, the number of common operational taxonomic units found between the mothers and children increased as the children aged. Oral microbiota of children was more like their mothers’ with increasing age, regardless of whether the mothers had dental caries. Mothers with dental caries may have a greater influence on the oral microbiota of children’s than those without dental caries as children age. The inclusion criteria for the exposure group were as follows: (1) mothers that had participated in the previous project; (2) mothers with one or more untreated dental caries, and without missing or filling teeth when their infants were12 months old; (3) healthy infants without hereditary diseases or deformities; (4) existing tooth eruption in the infant; (5) mothers and their infants without intake of antibiotics in the previous 2 months; and (6) infants without prior history of dental treatments. The inclusion criteria for those in the control group were the same as for those in the exposure group except the mothers had to be caries-free when the infants were 12, 18, and 24 months old (same criteria for 1 and 3-6).The sample size calculation was as following: the main outcome variable of the present study is the Shannon index when children were followed up to 24 months, because the Shannon index is the most commonly used index to assess microbial diversity. Through literature review and pilot study, it is assumed that the standard deviation of the Shannon index is 1.1, and the mean difference of Shannon index between the exposed group and the control group is 0.6 in a two-sided 5% significance level test with 80% power, each group requires 6 children. According to the two-year loss to follow-up rate is 20%, each group requires at least 8 children finally. A total of 20 pairs of children (aged 12 months) and mothers were included in this study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University (Project identification code: 201410).Written informed consent was obtained from all the parents of the children.For the clinical oral examination of the mothers and children , the participants’ dental caries and oral hygiene statuses were assessed. The mothers’ dental caries statuses were diagnosed by a trained and licensed dentist according to the criteria recommended by the World Health Organization (WHO) . Dental The dental plaque biofilms were collected to extract genomic DNA. After fasting for 2h, supragingival plaques were sampled from all the mothers’ and children’ teeth at 12, 18, and 24 months of age using a sterile curettage. Pooled plaque samples were immediately placed in a sterile Eppendorf tube with 1 ml of sodium thiosulfate solution, and then preserved at -80°C.Total genomic DNA of the dental plaque biofilms were extracted using the QIAGEN QIAamp DNA mini kit . The concentration and purity of the DNA was evaluated using a Nanodrop 2000 ultra-micro spectrophotometer . The variable regions (V4-V5) of 16S rDNA were amplified using universal primers 515f (5’-GTGCCAGCMGCCGCGG-3’) and 907Rb (5’-CCGTCAATTCMTTTRAGTTT-3’). About 10-50 ng of DNA was used for PCR-amplification, and the PCR reaction conditions were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 45s, annealing at 50°C for 1 min, elongation at 72°C for 1.5 min, and a final extension step at 72°C for 10 min. The PCR products were purified using the Gel Extraction Kit . The quality and the purity of the DNA were evaluated using the Quant-iTPicoGreen dsDNA Assay Kit and the Microplate reader , respectively. The 16SrRNA library was prepared using the TruSeq Nano DNA LT Library Prep Kit (Illumina) and sequenced with 5% PhiX samples using Illumina MiSeq sequencers with 600 cycles.Quality control was performed on the raw data in the FASTQ format by removing the reads with an average quality of < Q20, a length < 150 bp, and chimera and ambiguous bases. Two high-quality paired reads were recombined using the FLASH software (version 1.2.7)  to obtaiThe operational taxonomic units (OTUs) with 97% similarity were identified and classified using the taxonomy classification methods in the UCLUST software . The lonvia the Chao1, ACE, Shannon, and Simpson indices. Chao1 and ACE were used to estimate the community richness  . An analDifferences of questionnaire and oral clinical examination data between two groups were assessed by Mann–Whitney test and Bonferroni adjustments were used for multiple testing. One-way permutational multivariate analysis of variance (PERMANOVA) was calculated in QIIME software. All statistical analysis were produced using the R package. A P < 0.05 was considered to be significant.A total of20 pairs of mothers and their children aged 12 months were enrolled in this study. One pair was lost to follow-up at 18 months and one at 24 months  due to the nonlocal residence change. Therefore, 18 pairs completed all three follow-ups. As shown in At 12 months of age, all children had eruptions of only deciduous anterior teeth . At 18 months of age, one child in CEG group had developed white spot lesions on two teeth . At 24 months of age, two children in CEG group had developed white spot lesions on three teeth . During the follow-ups, 54 samples of supragingival plaque biofilms were obtained from the mothers and their children.OTU clustering and sequence annotation were conducted based on the sequences at a 3% dissimilarity level, and the OTU tables obtained were used for subsequent analyses. The OTU distributions for mothers  and children  at 12, 18, and 24 months, respectively, are shown in The alpha diversity indices, which included Chao1, Shannon, Simpson, and ACE, are shown in To analyze the microbial community structure of all the samples, a PCA was performed at the genus level, and the results were further compared between the groups through an Adonis/PERMANOVA analysis. The PCA showed that most of the samples in each group were clustered, and that most samples in M18, M24 and C24,the MEG24 and CEG24, and the MCG24 and CCG24 were clustered together Figure 3Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria were the dominant bacterial phyla, accounting for more than 80% of each group. The top six dominant genera in M12 were Prevotella, Fusobacterium, Capnocytophaga, Neisseria, Corynebacterium, and Selenomonas; the top six in C12 were Capnocytophaga, Neisseria, Streptococcus, Kingella, Leptotrichia, and Lautropia; that in M18 were Streptococcus, Neisseria, Haemophilus, Rothia, Burkholderia, and Porphyromonas; that in C18 were Selenomonas, Prevotella, Leptotrichia, Veillonella, Neisseria, and Streptococcus; that in M24 were Streptococcus, Neisseria, Haemophilus, Rothia, Burkholderia, and Porphyromonas; and that in C24 were Streptococcus, Leptotrichia, Neisseria, Selenomonas, Burkholderia, and Prevotella.At the taxonomic level, the microbial compositional ratios of the mothers and children at the three time points are shown in Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria were the dominant phyla. The top three dominant genera in the MEG12 group were Prevotella, Fusobacterium, Corynebacterium, Leptotrichia, and Campylobacter; that in the CEG12 group were Capnocytophaga, Neisseria, Lautropia, Streptococcus, Kingella, and Leptotrichia; that in the MCG12 group were Prevotella, Fusobacterium, Neisseria, Capnocytophaga, Selenomonas, and Corynebacterium; that in the CCG12 group were Neisseria, Capnocytophaga, Streptococcus, Kingella, Leptotrichia, and Veillonella; that in the MEG24 group were Streptococcus, Neisseria, Haemophilus, Burkholderia, Rothia, and Leptotrichia; that in the CEG24 group were Streptococcus, Burkholderia, Neisseria, Leptotrichia, Haemophilus, and Veillonella; that in the MCG24 group were Streptococcus, Neisseria, Haemophilus, Rothia, Burkholderia, and Porphyromonas; and that in the CCG24 group were Leptotrichia, Neisseria, Selenomonas, Streptococcus, Prevotella, and Capnocytophaga.In addition, the microbial relative abundance in the MEG and CEG at 12 months and 24 months are shown in Bacteroidia, Prevotellaceae, and Prevotella, while the children tended to have a higher relative abundance of Proteobacteria, Betaproteobacteria, and Neisseriales. In the 18-month group, mothers had a higher relative abundance of Proteobacteria, Bacilli, and Streptococcus, while the children had a higher relative abundance of Clostridia, Selenomonas, and Firmicutes. In the 24-month group, mothers had a higher relative abundance of Actinomycetales, Actinobacteria, and Rothia, while children had a higher relative abundance of Clostridia, Firmicutes, and Leptotrichia.The linear discriminant analysis effect size (LefSe) method was used to compare the enriched taxa in each group Figure 5Bacteroidia, Prevotellaceae, and Prevotella and in the CEG12,Proteobacteria, Betaproteobacteria, and Neisseriales were relatively more abundant. In the MCG12, Bacteroidia, Prevotellaceae, and Prevotella were relatively more abundant and in the CCG12, Neisseriales, Neisseria, and Bacilli had a higher relative abundance. Moreover, Actinomycetales, Actinobacteria, and Micrococcaceae were relatively more abundant in the MEG24, while Veillonella, Xanthomonadales, and Stenotrophomonas had a higher relative abundance in the CEG24. There was a higher relative abundance of Actinomycetales, Actinobacteria, and Rothia in the MCG24 and Fusobacteriales, Fusobacteria, and Leptotrichia had a higher relative abundance in CCG24. Notably, there were fewer differences in taxa between the MEG24 and CEG24 compared to that between the other three comparison groups. Additionally, the children had more enriched taxa than the mothers when the children were 24 months old.In the MEG12, there was a higher relative abundance of Dental caries is a complex multifactorial disease. A recent heritability study based on larger families reported that the heritability of dental caries in primary dentition is more than 50% . AdditioAt a 3% divergence level, there were 516, 725, and 817 OTUs in common between mothers and children at 12, 18, and 24 months of age, respectively. Additionally, 405 and 654 OTUs were identified in common between the MEG and CEG at 12 months and 24 months, respectively. A total of 390 and 717 OTUs were obtained in common between the MCG and CCG at 12 months and 24 months, respectively. The number of common OTUs increased with time, indicating that the children’s oral microbiota were more similar to their mothers with increasing age, regardless of whether the mothers had dental caries. It has been reported that an infant’s immune system is immature, and the plaque ecosystem evolves and matures gradually , which mThe oral microbial diversity in each group was compared using alpha diversity indices, including Chao1, Shannon, Simpson, and ACE. Comparison results showed that the alpha diversity indices in the mother groups were significantly higher than that in the children groups at 12 months, but there were no significant differences between the mothers and children at 18 and 24 months. Similar results were found in the mothers and children with or without caries, namely, the alpha diversity indices in the MEG12 were significantly higher than that in the CEG12.Additionally, the Chao1, Shannon, and ACE in the MCG12 were significantly higher than that in the CCG12. Moreover, the overall similarity between microbial communities in each group was determined by PCA. The results showed that the community composition in most of the children’s samples differed from that of the mothers at 12 months regardless of whether the mothers had caries. Taken together, these results indicated that alpha diversity indices and PCA analyses were consistent when assessing for microbial diversity. The oral microbiota of the children was more similar to that of their mothers with increasing age, regardless of whether the mothers had dental caries.Mother, who generally play as the main caregivers, would exhibit primary influence in the formation, transmission, and environmental conservation and social development of her children . Given tActinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria . Additionally, there were fewer differences in taxa between the MEG24 and CEG24 compared with that between the other three comparison groups. These findings suggest that mothers with dental caries may have a greater influence on their children’s oral microbial communities than those without dental caries as the children age. Studies have reported that a vertical transmission of cariogenic bacteria does exist between mother and child . ImportaPrevotella, Fusobacterium, Capnocytophaga, Streptococcus, Neisseria, and Haemophilus were found to be the dominant genera in the MEG groups. Streptococcus, Neisseria, Porphyromonas, and Prevotella have been reported to be strongly associated with dental caries  BioProject, The studies involving human participants were reviewed and approved by Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University (Project identification code: 201410). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Investigation: DF and DT. Methodology: HL. Project administration: XF. Supervision: MW and HL. Writing - original draft: FL. Writing - review and editing: WX. All authors contributed to the article and approved the submitted version.This study was supported by grants from the General Project of Shanghai Municipal Health Committee (Project No. 201940041), Shanghai Municipal Key Clinical Specialty (Project No. shslczdzk01601), and Shanghai Clinical Research Center for Oral Diseases (19MC1910600).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nocturia is a common and highly troubled lower urinary tract symptom, which has a wide range of effects. About 33% of patients with lower urinary tract symptoms have been affected by nocturia. Nocturia is mainly manifested as the increase of urination frequency and urine volume at night. It has been proved that acupuncture can reduce the symptoms of nocturia and regulate bladder function in Western countries. Acupuncture may be a promising choice for the treatment of nocturia.RCTs of acupuncture for nocturia will be searched in the relevant database, including PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure Wanfang Database, Chinese Biomedical Literature Database, and Chinese Scientific Journal Database. The studies of electronic searches will be exported to EndNote V.9.1 software. We will run meta-analyses using the Review Manager (RevMan) V.5.3 software. Any disagreement will be solved in consultation with a third reviewer.Our study aims to explore the efficacy of acupuncture for nocturia and to provide up-to-date evidence for clinical of nocturia.The conclusion of this study will provide evidence for the efficacy of acupuncture treatment of nocturia.INPLASY202130100. A retrospective study of the prevalence of nocturia from 1990 to 2009 found that among young adults aged 20 to 40 years, the incidence ranged from 11% to 35.2% in males and 20.4% to 43.9% in females, while the prevalence ranged from 68.9% to 93% in older adults aged 70 years and older.Nocturia is a common and highly troubling condition of the lower urinary tract characterized by increased frequency and volume of urination at night. Its occurrence and development are closely related to bladder hyperactivity and lower urinary tract symptoms, The essence is the result of an imbalance between functional bladder storage capacity and urine production. Nocturia is not only characterized by increased nocturnal urination, but also may be associated with the risk of other diseases. Frequent nocturnal awakenings due to nocturia can lead to worse sleep, increased daytime sleepiness, and reduced energy and activity in older adults. Older adults are also at increased risk of falling due to repeated night time bathroom visits, Datas show that, 25% of falls in the elderly are directly related to nocturia, and patients who use the night time bathroom at least two or more times per night more than double the risk of fractures and fall-related trauma. Nocturia is also associated with an increased risk of urinary tract disorders such as urethral obstruction, bladder diverticulum, hydronephrosis, and vesicoureteral reflux, as well as an increased prevalence of chronic diseases such as hyperglycemia, diabetes, coronary heart disease, and nervous system.–11 It is accompanied by a variety of complications seriously affect the physiological and psychological state of patients, reduce the quality of life (N-QoL) of individuals. Currently, nocturia is mainly treated with drugs, including desmopressin, α1 receptor antagonists, 5α reductase inhibitors, and antimuscarinic drugs.,14 These drugs mainly improve nocturia symptoms by enhancing the kidney's reabsorption of water during the night, regulate the activity of the brainstem urination center and improve urodynamic However, drugs often have side effects during treatment. For example, alpha 1 blockers can lead to cardiovascular disease, aggravate heart failure, induce vasodilation-induced lowering of blood pressure, lead to postostatic hypotension, and cause dizziness and falls. Desmopressin tends to cause hyponatremia, and 5-ARI can worsen erectile dysfunction and lower testosterone levels in men. Although some therapeutic measures such as lifestyle regulation, Magnetic stimulation, and botulinum toxin A (BoNT/A) have been used to promote the improvement of nocturia symptoms,–23 the efficacy of these treatments is relatively limited and there is A lack of efficacy data, so the treatment of nocturia still needs to be further improved.According to the pathophysiological mechanism of nocturia, its etiology is mainly caused by 4 aspects: nocturnal polyuria, 24-hour polyuria, decreased bladder volume and sleep disturbance. and sacral nerve regulation through acupuncture and moxibustion treatment, forming electrical nerve effect, so as to play a therapeutic role. At present, acupuncture has been widely used in the treatment of nocturia. An increasing number of clinical studies have shown that acupuncture can reduce the number of nocturia and nocturnal urine volume, and help improve patients’ bladder function and lower urinary tract symptoms.–31 Although there are some studies on acupuncture and moxibustion in the treatment of nocturia, the meta-analysis of acupuncture in the treatment of nocturia is still relatively blank. Therefore, we will use all relevant randomized controlled trials (RCTs) data for meta-analysis to comprehensively evaluate the clinical efficacy of acupuncture and moxibustion in the treatment of nocturia.Acupuncture, as a traditional Chinese medicine therapy, is a kind of nerve stimulation technology. In clinical practice, it can produce percutaneous tibial posterior stimulation2The aims are:1.to explore the efficacy of acupuncture for nocturia and2.to provide up-to-date evidence for clinical of nocturia.33.1,33 This protocol has been registered on INPLASY (registration number: INPLASY202130100: https://inplasy.com/inplasy-2021-3-0100/).The protocol for this review was developed in accordance with the PRISMA-P guidelines and the Cochrane Handbook.3.23.2.1All RCTs reported will be included without regional and language restrictions. Animal studies, cohort studies, case-controlled studies, case reports, and expert experience will be excluded.3.2.2 “the complaint that the individual has to wake at night 1 or more times to void” regardless the age, gender, race, country, and nocturia type.Participants in the study were diagnosed with nocturia. On the basis of International Continence Society -2002 report on standardization of terminology in nocturia3.2.3The purpose of this study is to observe the clinical study of acupuncture in the treatment of nocturia. Acupuncture treatment were used in the experiment, including body acupuncture, warm acupuncture, electro-acupuncture, auricular acupuncture, fire needling, elongated needle, moxibustion, or herbs-partitioned moxibustion.3.2.4The control group that will include nonacupuncture techniques, such as behavioral therapy, sham acupuncture, placebo, or pharmacotherapy. The acupoint numbers, retaining time, and frequency will not be restricted in this protocol.3.2.53.2.5.1The primary outcome measures will be total and frequency of nocturnal urination.3.2.5.2Secondary outcomes will include the ratio of nocturnal urine volume to daytime urine volume. Urine/blood osmotic pressure ratio, urine specific gravity value. change in N-QoL, Pittsburgh Sleep Quality Index Scale, as well as a standard battery of blood and urine analyses, vital signs, and physical examinations.3.3Non-RCTs;None of the valid outcome indicators;Duplicated data;Invalid outcome indexes.3.43.4.1RCTs of acupuncture for nocturia will be searched in the relevant database, including PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure Wanfang Database, Chinese Biomedical Literature Database, and Chinese Scientific Journal Database. The key words include “acupuncture,” “nocturia,” “complementary therapy,” “randomized controlled trial,” “randomized controlled trial” and “systematic review.” An equivalent translation of the same search terms will be used to search in the Chinese databases. The search strategy of PubMed is shown in Table 3.4.2A review or meta-analysis of relevant RCT systems will be conducted via electronic search. We will also manually search the references of relevant articles that is not included in the electronic database to further identify eligible studies.3.5The studies of electronic searches will be exported to EndNote V.9.1 software. Two authors will independently undertake the process of selecting the search results according to the inclusion and exclusion criteria. They will review and screen the titles and abstracts retrieved by literature search to exclude irrelevant trials. The causes of both selections will be documented and full texts will be obtained and checked for further evaluation if necessary. When there is uncertainty about eligibility of the study, reviewers will arrive at a decision by via discussion and consensus with a third reviewer. The selection process will be showed in a PRISMA flow diagram , moderate heterogeneity (30%–60%), substantial heterogeneity (50%–90%), and considerable heterogeneity (75%–100%). A random-effect model or subgroup analysis should be used when there exists significant heterogeneity.Statistical heterogeneity should be evaluated by Chi-Squared tests and 3.11I2 < 40%, the fixed-effects model will be used for data synthesis and analysis; If I2 ≥ 40% and < 75%, the random-effects model will be implied; If I2 ≥ 75%, it means there is considerable heterogeneity between studies. Alternatively, we will eliminate low-quality research and use sensitivity analysis to investigate which studies will be the most likely have a significant effect on heterogeneity. If synthesize quantitatively is not possible, we’re going to do a qualitative description.We will run meta-analyses using the Review Manager (RevMan) V.5.3 software. If the result of heterogeneity in 3.12If there is significant heterogeneity in the results, we will conduct a subgroup analysis to investigate differences in age, gender, length of disease duration, outcome style, etc.3.12.1We will perform sensitivity analyses to verify robustness of results. It includes the impact of methodological quality, study design, and sample size.3.13Two reviewers will independently use the Grading of Recommendations Assessment, Development and Evaluation, scored each criterion as having high risk, low risk or unclear risk.3.14The study will be published in peer-reviewed journals or relevant conferences. Ethical approval is not required. The results of this study will provide potential guidance for promoting the treatment strategy of nocturia patients.4 Many beneficial explorations on nocturia have been made around the world, which have made many important contributions to the understanding of etiology and pathology of nocturia as well as the prevention and treatment of the disease, and provided a variety of options for improving the N-QoL of patients with nocturia. However, due to the characteristics of recurrent nocturia and lingering difficulty in healing, the current treatment is difficult to achieve long-term good control of the symptoms of patients, and the safety of its prognosis and efficacy is still not ideal. Therefore, there is an urgent need for a stable, safe, and convenient treatment. Therefore, there is an urgent need for a stable, safe and convenient treatment. Acupuncture, as a traditional Chinese medicine therapy, is also increasingly widely used in the treatment of nocturia. Several RCT studies recently have also provided relevant evidence to estimate the effectiveness of acupuncture for treating patients with nocturia. But now is still lack of enough according to support to get a clear conclusion, therefore this study for the first time to a systematic review and meta-analysis of available literature, objectively evaluate the clinical efficacy of acupuncture in the treatment of nocturia, acupuncture treatment for the future nocturia provide objective statistics, and for clinicians treating nocturia increased reliable reference, benefit the patients.The frequent nocturnal urination caused by nocturia can negatively affect patients’ sleep quality, mental state, and N-QoL.Conceptualization: Yingjie Nie, Yushan Fan.Data curation: Yingjie Nie, Lilin Huang, Xiaojun Zhao.Formal analysis: Lilin Huang, Xiaojun Zhao, Ruikang Pang.Methodology: Ruikang Pang, Yijia Yang.Software: Xiaojun Zhao, Ruikang Pang, Yijia Yang.Supervision: Yushan Fan, Lilin Huang.Writing – original draft: Yingjie Nie, Ruikang Pang, Yijia Yang.Writing – review & editing: Yingjie Nie, Yushan Fan, Xiaojun Zhao.