Aims: The objective of this study was to assess the efficacy and potential mechanisms of Chinese herbal medicine (CHM) for treating coronary heart disease (CHD) patients with anxiety or depression.Methods: A systematic literature search was performed. Screening studies, extracting data, and assessing article quality were carried out independently by two researchers. The active ingredients of CHM for the treatment of CHD with anxiety or depression were analyzed by the network pharmacology, and the main potential mechanisms were summarized by the database of Web of Science.Results: A total of 32 studies were included. The results showed that compared with the blank control groups, CHM was more beneficial in treating anxiety or depression in patients with CHD , and the efficacy of CHM was not inferior to that of Western medicine (WM) . Additionally, CHM also showed a significant advantage in improving angina stability (AS) in CHD patients with anxiety or depression compared with blank groups  and WM groups . Angina frequency (AF) and electrocardiogram (ECG) analysis after using CHM demonstrated similar trends. Based on the network pharmacology, quercetin, kaempferol, luteolin, beta-sitosterol, puerarin, stigmasterol, isorhamnetin, baicalein, tanshinone IIa, and nobiletin were most closely and simultaneously related to the pathological targets of CHD, anxiety, and depression. The main underlying mechanisms might involve anti-damage/apoptosis, anti-inflammation, antioxidative stress, and maintaining neurotransmitter homeostasis.Conclusion: CHM exhibited an obvious efficacy in treating CHD patients with anxiety or depression, especially for improving the symptom of angina pectoris. The most active compounds of CHM could simultaneously act on the pathological targets of CHD, anxiety, and depression. Multiple effective components and multiple targets were the advantages of CHM compared with WM. Anxiety and depression are commonly found in patients with coronary heart disease (CHD), and the prevalence of CHD complicated with anxiety or depression is 21 and 13%, respectively . Percutavia which CHM treats CHD patients with anxiety or depression is still needed to be clarified. Therefore, by comprehensively analyzing published studies, a meta-analysis and systematic review were performed to assess the efficacy of CHM and the underlying mechanisms in the treatment of CHD patients with anxiety or depression, which might provide an essential clinical value for the disease management in the future.As an important treatment strategy, Chinese herbal medicine (CHM) is characterized by multiple components, multiple targets, and multiple channels. It has been verified that CHM had a satisfactory efficacy and fewer adverse effects on CHD with anxiety or depression . HoweverThis meta-analysis and systematic review were performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines .Published articles were searched comprehensively in electronic databases  up to October 2021. “Coronary disease OR coronary artery disease OR myocardial infarction OR acute coronary syndrome” AND “anxiety OR depression OR depressive disorder” AND “traditional Chinese medicine OR herbal medicine” AND “randomized controlled trial” and their common synonyms were used for the searching strategy. The detailed searching strategy was shown in supplementary material.The inclusion criteria of the articles were as follows: 1) all participants met the diagnostic criteria of CHD with anxiety or depression; 2) the number of subjects in each group was not less than 30; 3) CHD patients in control and trial groups received basic treatments, with antianxiety or antidepressant Western medicine (WM) used (WM groups) or not (blank control groups) in control groups, and oral CHM was used in trial groups; 4) Hamilton anxiety scale (HAMA) and Hamilton depression scale (HAMD) were used to evaluate patients’ anxiety and depression, respectively; and 5) the efficacy index of CHD included one of the following: ① electrocardiogram (ECG); ② angina stability (AS) and angina frequency (AF) come from Seattle Angina Questionnaire; and ③ traditional Chinese medicine syndrome (TCMS) score.The exclusion criteria of the studies were as follows: 1) Nonclinical study and irrelevant research; 2) CHM was used in control groups, antianxiety or antidepressant WM was used in trial groups; 3) articles with incomplete data; 4) articles more than one high-risk item; and 5) review, meta-analysis, and conference abstracts.Retrieved articles were assessed independently by two researchers (YT and SL) according to the inclusion and exclusion criteria. Data, including first authors’ name, year of publication, sample size, age, gender, diseases, therapeutic regimen, final duration of treatment, dosage form and compositions of TCM, and the outcome index, were extracted from the included studies. The CHM was reported in scientific name, not the Latin name in pharmacopeia to avoid confusion  Rivera . Any disTwo authors (YL and YT) independently assessed the methodological quality according to the Cochrane risk-of-bias tool . Suffici2 < 50%), the fixed effect model was selected to analyze the data; otherwise, a random-effects model was applied. The subgroups analysis was based on whether control groups used WM or not. Sensitivity analysis was performed to explore potential effect modification. Also, funnel plots were used to assess publication bias. p < 0.05 was considered statistically significant.RevMan 5.3 software provided by the Cochrane Collaboration was used for meta-analysis. The odds ratio (OR) and standard mean difference (SMD) were used to analyze the pooled effects of dichotomous outcomes and continuous variable, respectively. When the heterogeneity of included studies was low  were summarized by the database of Web of Science.A total of 2,102 records were identified from eight electronic databases. Thirty-two studies met the inclusion criteria, and 2070 studies were excluded due to 1) irrelevant studies; 2) nonclinical studies; 3) review, meta-analysis, and conference abstracts; 4) sample size was less than 30; 5) using WM in trial groups; 6) non-HAMA or HAMD for evaluating the efficacy of anxiety or depression; 7) non-ECG or AS or AF or TCMS score for evaluating the efficacy of CHD; and 8) articles with incomplete data or more than one high-risk item. The specific screening process is illustrated in Thirty-two studies included 15 studies on CHD with anxiety  and 17 sFor control groups of CHD with anxiety, four studies used flupentixol and melitracen tablets , diazepaThe study methodological quality is concluded in As shown in p < 0.00001, I2 = 0%] , p = 0.52, I2 = 67%]. Moreover, a repetitive meta-analysis by consecutively excluding each study in WM groups was performed. The study by Qi et al. was the main source of heterogeneous, but it was not removed because of reasonable research design.In I2 = 0%] , and thep = 0.0002, I2 = 0%] , p = 0.002, I2 = 0%] , I2 = 0%] . In addiI2 = 0%] .p < 0.00001, I2 = 45%; AF: SMD = 0.71, 95%CI , p < 0.0001, I2 = 64%] , p < 0.0001, I2 = 62%], and WM groups  , 2 = 64%] , 5, blanI2 = 0%] , 5.As shown in p = 0.0003, I2 = 0%] , p = 0.18, I2 = 33%] , I2 = 0%] . The res2 = 33%] .p = 0.004, I2 = 0%] , p = 0.10, I2 = 0%] , I2 = 0%] . In addiI2 = 0%] .p < 0.00001, I2 = 0%; AF: SMD = 11.13, 95%CI , p < 0.00001, I2 = 6%] , p < 0.0001, I2 = 0%; AF: SMD = 10.34, 95%CI , p < 0.0001, I2 = 48%] , I2 = 6%] , 9, blan2 = 48%] , 9.Bupleurum chinense DC. , Glycyrrhiza uralensis Fisch. ex DC. , Ligusticum chuanxiong Hort. , Salvia miltiorrhiza Bunge , Angelica sinensis (Oliv.) Diels , Paeonia lactiflora Pall. , Pinellia ternata (Thunb.) Makino , Curcuma aromatica Salisb. , and Citrus×aurantium L.  were commonly used for treating CHD with anxiety or depression  were searched in the Web of Science database. As shown in β/6, tumor necrosis factor-α, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin, or elevating the IL-10 level in CHD patients. The anti-inflammatory role of quercetin, puerarin, or nobiletin was reported in anxiety or depression. Additionally, almost all active ingredients except stigmasterol and nobiletin possessed the functions of antioxidative stress and balancing level of reactive oxygen species, malondialdehyde, myeloperoxidase, and catalase, superoxide dismutase, glutathione. Quercetin, tanshinone IIa, and nobiletin were reported to reduce the levels of α-smooth muscle actin, angiotensin II, collagen I/III, matrix metalloproteinases 2/9, transforming growth factor (TGF)-β, and Smad7 to prevent myocardial fibrosis, which was one of the complications associated with myocardial infarction. Besides, the imbalance of adrenocorticotropic hormone, 5-hydroxytryptamine, brain-derived neurotrophic factor (BDNF), acetylcholine, noradrenaline, dopamine, and gamma-aminobutyric acid, which caused anxiety or depression, could be regulated by quercetin, kaempferol, luteolin, beta-sitosterol, puerarin, stigmasterol, baicalein, tanshinone IIa, or nobiletin. Isorhamnetin and baicalein could improve the depression by inducing neuronal differentiation and protecting synaptic plasticity, respectively. The roles of active compounds in regulating autophagy and improving mitochondria were also reported. Overall, the related mechanisms of TCM-active compounds in treating CHD with anxiety or depression contained a variety of signaling pathways, such as nuclear factor-kappa B, mitogen-activated protein kinase, Jun N-terminal kinase, extracellular signal-regulated kinase1/2, signal transducers and activators of transcription3, TGF-β1/Smad3, phosphatidylinositol 3-kinase/protein kinase B, and BDNF.The related mechanisms of these top 10 active ingredients in CHD, anxiety, and depression are summarized in There is accumulating evidence showing high prevalence of anxiety and depression comorbidities in patients with CHD. SSRIs and benzodiazepines are frequently used for treating depression or anxiety disorders, and the effectiveness of these drugs on psychiatric disorders has also been acknowledged . HoweverTCM has been reported to be effective in treating CHD, anxiety, and depression with a less adverse effect, and might be a potential therapeutic option for patients with subsyndromal anxiety or depression. However, the efficacy and benefit of CHM in treating CHD with anxiety or depression still need to be further verified due to poor methodological quality and potential confounding factors. This meta-analysis and systematic review was performed to provide the evidence for the application of CHM in CHD patients with anxiety or depression. Thirty-two studies  were included for the evaluation of the efficacy of CHM. The results showed that CHM had a significant benefit on anxiety and depression in CHD patients, and its efficacy was not inferior to that of WM. Importantly, CHM also had a significant advantage to alleviate the angina symptom compared with blank control and WM groups. Besides that, there were no obvious adverse effects of CHM in the included studies .Bupleurum chinense DC. , Glycyrrhiza uralensis Fisch. ex DC. , Ligusticum chuanxiong Hort. , Salvia miltiorrhiza Bunge , Angelica sinensis (Oliv.) Diels , Paeonia lactiflora Pall. , Pinellia ternata (Thunb.) Makino , Curcuma aromatica Salisb. , and Citrus×aurantium L.  were commonly used for CHD with anxiety and depression. The phytochemicals identified in the CHM could act on the pathological targets of CHD, anxiety, and depression simultaneously.Furthermore, the frequency of CHM used in the included studies was analyzed, and the commonly used drugs were analyzed by network pharmacology. The results concluded that the CHM regulating Qi and promoting blood circulation, including Inflammatory response to vascular injury participates in the pathological processes of the atherosclerosis and CHD, and is associated with the increased risk of cardiovascular events and recurrent myocardial infarction . OxidatiOverall, these findings reveal that the CHM has a satisfactory efficacy for CHD with anxiety and depression, especially for improving the symptom of angina pectoris. Of importance, CHM itself contains multiple components that play critical functions in a large number of signaling pathways involved in distinct biological processes of CHD with anxiety or depression mainly including anti-damage/apoptosis, anti-inflammation, antioxidative stress, and maintaining neurotransmitters homeostasis. Compared with WM’s single effect on the nervous system, CHM may extert its functions in multiple places and systems by targeting distinct factors in CHD with anxiety or depression to improve both CHD and anxiety/depression syndromes.First, the sample size in each group of included studies was not more than 50, except the study by CHM had a significant efficacy for the treatment of CHD patients with anxiety or depression. Particularly, CHM could improve the symptoms of angina pectoris while alleviating anxiety and depression. The main mechanisms underlying the functions of these CHM-active ingredients might involve anti-damage/apoptosis, anti-inflammation, antioxidative stress, antifibrosis, maintaining neurotransmitters homeostasis, and regulating autophagy.


To develop and make available, at no cost to the user, Information and Communications Technology (ICT) tools for Dentistry, providing dental information and advice geared toward patients undergoing orthodontic treatment with fixed appliances.A Dentistry-based content that contemplated information and advice concerning orthodontic treatment with fixed appliances was elaborated. The materials, which included instructions on oral hygiene and treatment strategies when faced with possible complications, were evaluated and validated by specialists, whose assessments reached a 85% approval. From the validated content, products using four distinct ICT tools were formulated.The following technological products were developed: a program for community radios, three blog posts, four educational and informative videos, and a smartphone application - using texts, as well as images and videos. These ICT tools, geared toward patients wearing fixed orthodontic appliances, were made available by internet at no cost to the user, and the number of accesses is already expressive.These technological-scientific tools, developed and provided freely to the population, can aid patients during their treatment with fixed orthodontic appliances, contributing to the dissemination of reliable information, and clarifying doubts that may arise during orthodontic therapy. These free ICT tools serve to facilitate access to scientific knowledge, thereby favoring social inclusion, bearing in mind that this educational and informative material was offered in a simple and accessible manner to the general population. In this context, and resisting to decades of change, radio programs continue to be a widely renowned communication strategy for social, cultural, and healthcare development of many communities.,Dental professionals need to instruct their patients, help them to understand the importance of the recommendations given, and motivate them to maintain treatment adherence. In practice, this need has proven to be particularly difficult in Orthodontics, due mainly to the young age of most patients, and to the long period of time required for treatment.,However, the variety of content related to Dentistry disseminated on the internet present a questionable quality of information, which is often unreliable.Pontifícia Universidade Católica de Minas Gerais (CAAE #11137119.7.0000.5137).Among a wide range of information and guidelines related to orthodontic treatment with fixed appliances - considering its pertinence, the greater amplitude provided, and thinking about the different steps of the process -, a content was created, based on scientific evidence, which contemplated guidelines including recommendations on patients’ collaborative behavior, instructions on oral hygiene, and procedures to be taken when faced with unfavorable orthodontic complications. The present study was approved by the Research Ethics Committee of In an attempt to make a truly appropriate content available, its validation was conducted by means of an analysis performed by a committee of specialists, Masters and Doctors in Orthodontics, members of the Brazilian Association of Orthodontics , who were lecturers or consultants in post-graduate programs in different universities. This process was based on a prior study, which reported that at least six specialists, called “judges”, should examine the following criteria:» Clarity of language: analyze if the textual content is clear, easily understandable, and appropriate for the target population.» Theoretical relevance: consider the association between content and theory.» Practical pertinence: evaluate if the content is appropriate for the public for which it has been created.®, were sent, via e-mail, to 10 possible judges. The answers for the questionnaire were constructed following the Likert scale, with five alternatives: 1) Strongly disagree, 2) Disagree, 3) Neither agree nor disagree, 4) Agree, 5) Strongly agree. In addition to the use of this scale, a space was also created for the judges to provide criticisms, suggestions, and recommendations, which is a common procedure in validation studies.,,Invitation letters, together with the elaborated material  and a virtual validation questionnaire , createdBearing in mind that the evaluation reached an average of 85% approval among the specialists , a new vFrom the validated material, ICT tools for Dentistry were developed, enabling the production of: one community radio program; three blog posts; four videos; and, considering the broad popularity of mobile devices with internet access, one smartphone application.The community radio program scripts were drafted  using thA smartphone application was also developed, seeking to surpass physical and management barriers of dental care. One prototype was designed, considering functionalities, layout, and content. Among the functionalities, features such as the patient’s intuitive navigation and easy interaction were added. The layout was idealized to be “simple” and “practical”, facilitating the navigation. As regards the provided information, the validated content was organized using not only texts, but also images and videos.The application was developed for the Android platform, which is an operational system that is highly compatible with smartphones of different brands. Tools made available by Google, such as the Android Studio development environment, were used. In addition to this environment, the Firebase development platform was also used, which is also provided by Google. The source code of the algorithms for the application was written in Java language. As user data were obtained, the SHA and MD5 algorithms  were also used. The graphics were generated in XML (Extensible Markup Language), producing a great capacity of interaction with the user.As the layout is the visual part of the system, menu items, buttons, and so forth were used, as is recommended by Google in its development documentation. The images and icons were generated according to the Google documentation specifications. The source project of the application was then stored in a closed repository in GitHub. Both the prototype and the development source code were registered at the National Institute of Industrial Property , and the name “OrthoHelp” was chosen for the application.During all stages of development of these technological tools, it was valued that, in addition to being reliable, the content should be accessible. In this way, all the tools were made available for free.The drafted and validated content was made available using the following ICT tools: radio, blog, videos, and smartphone application.“I’m undergoing orthodontic treatment with braces. What should I be doing in terms of oral hygiene?” .,,The content was also structured to attend to the specificities of a blog. In the field of health, blogs have become quite widespread as a means of communication since 2010.-,TM.TM about the specialty are weak. More accessible information, such as oral health care during orthodontic treatment, can be easily disclosed and shared by any user on YouTubeTM, and the fact that the information is often not provided by a dental professional confers a high probability of imprecision and unreliability to the recommendations provided.,,TM can improve the knowledge of the patient about the orthodontic treatment, when compared with information provided in leaflets or by verbal communication, and have the advantage of being able to be accessed at any time on any mobile device with internet connection, such as smartphones.The use of educational videos for health promotion has been investigated.,Mobile technology has become a common reality in daily life and has been influencing a new way of exchanging information and interactivity among its users. Smartphones and its applications have very quickly become accessible to the majority of the population, and the development of an application for mobile telephones in the field of health explores the possibility of crossing geographic and cultural barriers.Participants of a study that evaluated preferences related to the use of applications, such as educational aids for patients undergoing orthodontic treatment, reported that the applications for dental education should be used for a better communication between dentist and patient, and to increase the patients’ understanding of the dental procedures that they are undergoing.,,Notably, oral hygiene conducted in an appropriate manner can be used as an effective preventive measure against caries and to control periodontal disease and, even though it is a relatively simple action, it is constantly neglected.ICTs are widely used in health, even among the less favored social classes, and faced with this social relevance, the dissemination of reliable and precise information in oral health care needs to be encouraged and expanded.Technological products, provided at no cost to the users through specific free ICT tools, make it possible to facilitate the access to scientific knowledge, which is of utmost importance, especially in terms of social inclusion. In addition, they provide alternative and complementary means of education and encouragement of health care, which can contribute to the quality of life of patients undergoing treatment with fixed orthodontic appliances.By providing a wide range of technical tools created in this study , it is our intent to contribute to advances in the concession of information to the general population, fact that can be corroborated by the large number of accesses, downloads and views  to the content developed here. These tools can favor the patient’s understanding of the explanations provided by dentists, which can instill a greater peace of mind, security, and comfort during orthodontic therapy for both patients and dental professionals.


Bladder cancer has the characteristics of high morbidity and mortality, and the prevalence of bladder cancer has been increasing in recent years. Immune and autophagy related genes play important roles in cancer, but there are few studies on their effects on the prognosis of bladder cancer patients.Using gene expression data from the TCGA-BLCA database, we clustered bladder cancer samples into 6 immune-related and autophagy-related molecular subtypes with different prognostic outcomes based on 2208 immune-related and autophagy-related genes. Six subtypes were divided into two groups which had significantly different prognosis. Differential expression analysis was used to explore genes closely related to the progression of bladder cancer. Then we used Cox stepwise regression to define a combination of gene expression levels and immune infiltration indexes to construct the risk model. Finally, we built a Nomogram which consist of risk score and several other prognosis-related clinical indicators.The risk model suggested that high expression of C5AR2, CSF3R, FBXW10, FCAR, GHR, OLR1, PGLYRP3, RASGRP4, S100A12 was associated with poor prognosis, while high expression level of CD96, IL10, MEFV pointed to a better prognosis. Validation by internal and external dataset suggested that our risk model had a high ability to discriminate between the outcomes of patients with bladder cancer. The immunohistochemical results basically confirmed our results. The C-Index value and Calibration curves verified the robustness of Nomogram.Our study constructed a model that included a risk score for patients with bladder cancer, which provided a lot of helps to predict the prognosis of patients with bladder cancer.The online version contains supplementary material available at 10.1186/s12863-022-01073-7. Bladder cancer (BLCA) is a high-incidence tumor which has high morbidity and mortality. According to statistics, BLCA ranks ninth in the prevalence of malignant diseases and 13th among the most common causes of cancer death [For the past decades, BLCA has made many advances in clinical treatment. Gene sequencing technology has identified the most mutated genes in BLCA. Many studies have researched the treatment of BLCA at the level of cells and molecular mechanism . MeanwhiMost of the immunomodulatory effects of immunotherapy are achieved by enhancing T cell responses. Although immunotherapy has brought many new breakthroughs to the treatment of tumors, only a small proportion of patients profit from it, which highlight the necessity of identifying new cells and new molecules , 5. For In many cancers, the regulations of autophagy play important roles. Autophagy plays dynamic inhibitory or promotion effects in different stages of tumors. Therefore, understanding how autophagy regulates metabolism and tumor growth is essential for tumor treatment , 8.In recent years, methods of bioinformatics and data mining have been increasingly applied to various medical researches. In the field of oncology, researchers have achieved many clinically meaningful results using these methods. Xie et al. found and confirmed the significant role of ITPA in uveal melanoma through bioinformatics analysis and cell experiments, which provided a certain reference for the diagnosis and treatment of uveal melanoma .In the present study, immune genes and autophagy genes were collected. We utilized gene expression data from two commonly used databases (The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO)) to construct BLCA molecular subtypes based on immune-related and autophagy-related genes. It showed that these subtypes distinguished well among patients with different prognosis of BLCA , 11. Thrhttps://portal.gdc.cancer.gov/). The gene expression data consisted of 166, 116 BLCA samples from GSE13507 and GSE48276 datasets were downloaded from GEO database (http://ncbi.nlm.nih.gov/geo/), respectively. We conducted preliminary screening of the downloaded original data according to the following criteria: (1) genes with zero expression in more than 30% of the samples were excluded; (2) excluding the genes whose expression values were lost; (3) excluding the samples without related clinical data; (4) excluding the samples with OS < 30 days; (5) excluding non-tumor tissue samples. We divided the TCGA-BLCA dataset into two random cohorts according to the ratio of 3 to 1: training cohort (n = 307) and internal validation dataset (n = 102). GSE13507 and GSE48276 datasets were regarded as external validation cohorts. Then, we downloaded 2208 human immune genes and autophagy genes from the Reactome database (https://reactome.org/) [The TCGA-BLCA dataset included the RNA-seq data of 410 BLCA and 37 adjacent normal samples was downloaded from the TCGA database (me.org/) .Based on the 2208 genes selected above, those screened training cohort samples were clustered according to the non-negative matrix factorization (NMF) in R package “NMF”. Moreover, we used Cibersort to calculate the immune infiltration scores of the subtypes for molecular subtype analysis .P < 0.05 and FDR < 0.25).We utilized the R package “DESeq2” to compute the differentially expressed genes (DEGs) according to the criteria FDR < 0.05 and FC > 1.5. Gene Ontology (GO) Term Enrichment Analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Analysis were conducted based on the DEGs –16. MoreP < 0.05 was considered significant statistically. Then, we utilized Cox stepwise regression to further narrow the variables obtained above. Finally, a risk score model including variables weighted by their Cox stepwise coefficients was established. According to the formula: risk score = h0(t)*βvariable(n)), we figured up the risk score for each sample, separately. A TCGA-BLCA internal verification cohort and two GEO external verification cohorts were used to verify the credibility of the model, respectively. In order to further evaluate the robustness of the model, we used the R package of “pROC” to plot the risk score distribution of each cohort according to the risk score of each sample. According to the different risk scores of samples, samples with scores greater than zero were put in high-risk group, samples with scores less than zero were put in low-risk group. The Kaplan-Meier (KM) survival curve was used to perform the OS of each group and the log-rank test was used to compare the survival differences between the samples of high-risk group and low-risk group.We used above DEGs expression data to build a risk score model. Univariate Cox survival analysis in R studio was used to analyze the correlation between each DEG, immune infiltration score and OS of the BLCA patients. Wald test P < 0.001). We analyzed the pTNM staging of patients in high-risk group and low-risk group. After excluding patients without pTNM staging and TX, NX, and MX staging, we found no significant difference in pTNM staging between the two groups. (P > 0.05) (Table SP < 0.01)  Table S. T cellsStaphylococcus aureus infection . Then, we used the ROC curve to test the prediction efficiency of the risk model. Fig. P < 0.05). Except that the influences of the expression levels of FBXW10 and IL10 on prognosis were inconsistent with our conclusion, the other results above all confirmed our view.According to the training cohort of TCGA-BLCA dataset, the risk model was constructed. By performing univariate Cox regression analysis for each variable, we found that a total of 29 variables were significantly associated with the prognosis of BLCA patients. To establish our risk model, Cox stepwise regression was used to further screen the variables. Finally, a model with 15 variables was selected. Risk score of each sample is shown in Table SP < 0.001), and the low-risk group was better than the high-risk group. Then, we drew ROC curve of the risk model, AUC of the model was 0.815 . Then we further performed Cox stepwise regression analysis on the above four risk factors, and the results showed that age, tumor stage and risk score were independent risk factors for OS. The nomogram was shown in Fig. We screened for prognostic risk factors from clinical information collected from the training cohort samples. After univariate Cox regression analysis, Age, tumor stage, N stage and risk score were found to be risk factors for OS  mediates multiple immune system functions, including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release . As a reGHR may be implicated in many types of cancer. Studies related to gastric cancer have shown that GHR regulated the G1 cell cycle progression by mediating the PI3K/AKT signaling pathway, thereby regulating the growth and apoptosis of gastric cancer cells . Strous IL10 is thought to have the ability to suppress antitumor T cell responses in cancer, but several researches have also suggested that IL10 took part in some inherent antitumor T cell responses. It indicates that IL10 may play a dual regulatory role in cancer . In our Studies related to chronic non-bacterial osteomyelitis indicated that the frequency of MEFV gene mutations increased in the disease, and the disease phenotype was more severe in patients with MEFV gene mutations . In addiLOX-1 encoded by the OLR1 gene is involved in the pathogenesis of atherosclerosis, and activation of LOX-1 is an important mechanism leading to plaque instability and progression to acute coronary syndrome . MeanwhiAt present, there are relatively few studies on PGLYRP3. According to research, PGLYRP3 acted a pivotal part in antibacterial immunity and inflammatory responses , 40. In According to research, RASGRP4 was significantly overxpressed in diffuse large B cell lymphoma. Meanwhile, knockdown of RASGRP4 significantly inhibited tumor formation . StudiesS100A12 was proved to be a useful biomarker in inflammatory conditions. And some studies suggest that it might also take part in cardiovascular disease . In cancImmunity and autophagy play important roles in tumors. Our study identified twelve genes associated with immunity and autophagy and three Cibersort immune infiltration scores that were significantly associated with bladder cancer prognosis. On this basis, we established a model to predict survival in patients with BLCA. There are some limitations to our study. First, the genes we defined were validated only by immunohistochemistry in the HPA database. Although the immunohistochemical data in the HPA database and the gene expression data in the TCGA are of relatively high quality, the data we used were from urothelial carcinoma and were not 100% representative of bladder cancer. Second, immunohistochemical information of FBXW10, MEFV, OLR1 and RASGRP4 were missing in HPA. The high expression of IL10 was detrimental to prognosis, which was inconsistent with our findings. Besides, the functions of these genes in bladder cancer need to be further explored. For BLCA patients with T1G3 stage, Bacillus Calmette-Guerin (BCG) treatment and response to BCG have important influence on the prognosis of patients. Unfortunately, our study did not have enough data at this point to make a credible statistical analysis, which was one of the limitations of this study. At the same time, genetic mutations may also have a significant impact on the prognosis of patients with bladder cancer. In many tumors, mutations in one or more genes have been shown to be significantly associated with prognosis. Unfortunately, we did not explore the genetic mutations in high-risk and low-risk patients.Additional file 1: Table S1. The difference of pTNM staging between high-risk group and low-risk group was significant.Additional file 2: Table S2. The difference of immune infiltration index between high-risk group and low-risk group was significant.Additional file 3: Table S3. 2208 immune-related and autophagy-related genes.Additional file 4: Table S4. Differentially expressed genes.Additional file 5: Table S5. GSEA KEGG enrichment.Additional file 6: Table S6. Risk score of each sample from the training cohort.Additional file 7: Table S7. Regression coefficient for each variable.


Kidney cancer is one of the most common urological cancers worldwide, and kidney renal clear cell cancer (KIRC) is the major histologic subtype. Our previous study found that von-Hippel Lindau (VHL) gene mutation, the dominant reason for sporadic KIRC and hereditary kidney cancer-VHL syndrome, could affect VHL disease-related cancers development by inducing telomere shortening. However, the prognosis role of telomere-related genes in kidney cancer has not been well discussed. In this study, we obtained the telomere-related genes (TRGs) from TelNet. We obtained the clinical information and TRGs expression status of kidney cancer patients in The Cancer Genome Atlas (TCGA) database, The International Cancer Genome Consortium (ICGC) database, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Totally 353 TRGs were differential between tumor and normal tissues in the TCGA-KIRC dataset. The total TCGA cohort was divided into discovery and validation TCGA cohorts and then using univariate cox regression, lasso regression, and multivariate cox regression method to conduct data analysis sequentially, ten TRGs  risk model had been constructed finally. The kidney patients in the high TRGs risk group represented a worse outcome in the discovery TCGA cohort (p<0.001), and the result was validated by these four cohorts . In addition, the TRGs risk score is an independent risk factor for kidney cancer in all these five cohorts. And the high TRGs risk group correlated with worse immune subtypes and higher tumor mutation burden in cancer tissues. In addition, the high TRGs risk group might benefit from receiving immune checkpoint inhibitors and targeted therapy agents. Moreover, the proteins NEK6, RF2, and ISG15 were upregulated in tumors both at the RNA and protein levels, while PLCL1 and PRKCQ were downregulated. The other five genes may display the contrary expression status at the RNA and protein levels. In conclusion, we have constructed a telomere-related genes risk model for predicting the outcomes of kidney cancer patients, and the model may be helpful in selecting treatment agents for kidney cancer patients. Kidney cancer is one of the most common urological cancers worldwide, and kidney renal clear cell cancer (KIRC) is the major histologic subtype , 2. The Telomeres are regions composed of repetitive TTAGGG DNA sequences and shelterin complex located at the end of chromosomes . TelomerMoreover, many studies have been conducted to investigate the role of telomeres in the development and progression of cancers. Recent findings suggest that the shortening telomeres can affect the process of cancer development in two different ways. First, telomere shortening might play a tumor-suppressive role by arresting cell proliferation. On the other hand, telomere shortening could also result in extensive genome instability, which promotes cancer progression. In breast cancer, long telomere lengths correlated with a better prognosis . A meta-Previous studies have focused on the telomere length in cancers and its role in the prognosis of cancers. There has been no study done to investigate telomere-related genes in the prognosis of cancers. Herein, we constructed a risk model using telomere-related genes to predict the prognosis of kidney cancer and then evaluated the potential role of this risk model in selecting treatment agents.http://www.cancertelsys.org/telnet/ (Kidney cancer patients’ data files from The Cancer Genome Atlas (TCGA) dataset (TCGA-KIRC cohort), The International Cancer Genome Consortium (ICGC) database (RECA-EU cohort), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) database (PDC000127) were downloaded and processed according to the operational processes of the public data provider. All the data in the TCGA-KIRC cohort were enrolled in this study to screen the differential expression genes. The analysis included the mRNA expression data and exact clinical features in all patients from these three datasets, including the survival time and tumor characteristics. The tumor mutation burden (TMB) data of the TCGA-KIRC cohorts were also downloaded and processed. In addition, the protein expression status of the CPTAC cohort and the immunohistochemical (IHC) data of renal cancer in the Human Protein Atlas (HPA) database were also acquired. In addition, the telomere-related genes were obtained from /telnet/ .The RNA sequence profiles of 533 kidney cancer tissues and 72 adjacent normal kidney tissues were used for screening the differential genes using the limma package . TelomerThe TCGA-KIRC cohort was randomly divided into the discovery TCGA cohort and the validation cohort in a 1:1 ratio. The discovery TCGA cohort was analyzed sequentially through univariate cox regression, Lass regression, and multivariate cox regression. Subsequently, the risk model was constructed based on the genes’ expression value and the coefficient, which were acquired in the multivariate cox regression using the Akaike Information Criterion (AIC) method. The risk score of each patient was calculated in these five cohorts: discovery TCGA cohort, validation TCGA cohort, total TCGA cohort, ICGC cohort, and CPTAC cohort. Each cohort was divided into two groups, the high-risk group, and the low-risk group, according to the median risk score, and the prognosis of each risk group was examined using the log-rank test. The risk score was also evaluated using univariate and multivariate cox regression to determine its role in predicting the overall survival of kidney cancer patients in the five cohorts. In addition, the receiver operating characteristic (ROC) curve was used to check the accuracy of the risk model in predicting the prognosis. Finally, a nomogram was constructed to predict the 1-, 3-, and 5-year survival rates using the risk score based on the total TCGA cohort.https://cibersort.stanford.edu/) (https://tide.dfci.harvard.edu/)  . The immrd.edu/) . Additiord.edu/) .The targeted drugs’ half-maximal inhibitory concentrations (IC50) were predicted using the gene expression level to reflect the treatment sensitivity. This was done using the R package named “pRRophetic” , 24.−ΔΔCt method and normalized with the beta-actin  and six RCC cell lines  were used to investigate the telomere-related gene expression status. Among these cell lines, 769-P, A498, 786-O, and OSRC-2 were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. While HK2, Caki-1, and ACHN were cultured in DMEM medium with 10% fetal bovine serum. TRIZOL reagent  was used to extract the total RNA, and the reverse transcription reactions were carried out using a qPCR RT Kit . Expression levels of telomere-related genes were detected by qRT-PCR). The human beta-actin gene was used as a reference gene. The primers were shown in ta-actin .The expression of proteins encoded by the genes included in the telomere-related genes model in the CPTAC cohort was compared using the limma package. In addition, the protein expression status of renal cancer and kidney tissues in the HPA database was accessed and analyzed using the “HPAanalyze” package . Continu2 = 0.84, P<0.001, The detailed characteristics of the three cohorts are summarized in the Among the 353 differential expression genes, 47 genes correlated with kidney cancer patients’ overall survival in the discovery TCGA cohort. Then 19 genes in these 47 genes were finally screened by Lasso regression. Finally, ten telomere-related genes  in these 19 genes were identified as independent risk factors through the multivariate cox regression and were used to format the risk model, telomere related genes (TRGs) risk model. And the TRGs risk score formula was as follows: risk score=0.005134398*ISG15 +0.047733021*RFC2-0.071026696*TRIM15-0.0203981*NEK6-0.133649782*PRKCQ-0.007396692*A TP1A1+0.700714514*ELOVL3+0.206037577*TUBB2B-0.148322187*PLCL1+0.070770091*NR1H3. The risk score of patients in five cohorts was computed, and the patients in each cohort were divided into a high and low-risk group according to each cohort’s median risk score value. The patients in the high-risk group represent the worse outcomes in all these five cohorts, discovery TCGA cohort (p<0.001), validation TCGA cohort (p<0.001), total TCGA cohort (p<0.001), ICGC cohort (p=0.018) and CPTAC cohort (p=0.003) 3. In adThe patients in the high-risk group showed a higher TMB (P=0.0023) than those in the low-risk group , and macrophages M0 had a higher rate of infiltration in the high-risk group than in the low-risk group. T cells CD4 memory resting, monocytes, macrophages M2, dendritic cells resting, and mast cells resting showed less infiltration in the high-risk group than in the low-risk group , C2 (IFN-g dominant), C3 (inflammatory), C4 (lymphocyte depleted), C5 , and C6 (TGF-b dominant) . The immThe TIDE score was significantly higher in the high-risk group than in the low-risk group  with a worse outcome than in the low-risk group (3%). In addition, the high-risk group showed a high TIDE score, indicating that the high-risk group patients may be more likely to experience an immune escape. Overall, the results suggest that the differences in prognosis between the high and low-risk groups might be partly due to the different immune statuses of the patients.High TMB has been identified as a biomarker to predict the potential benefit of immune checkpoint blockade (ICB) therapy. Our study showed that the high-risk group was associated with a high TMB . The dru+/K+ATPase. Studies have found that ATP1A1 mutations could cause aldosterone-producing adenoma (APA). Inhibiting ATP1A1 expression in pancreatic ductal adenocarcinoma (PDAC) cells can suppress the tumor invasion. In kidney cancer, the promoter methylation rate of ATP1A1 was about 15.8% . We have discussed the protein and RNA expression status of these genes in kidney cancer; however, there were no experiments to validate our findings. The model we have constructed may be helpful in the selection of treatment agents for kidney cancer patients.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found 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.Conceived and designed the experiment: X-HN, S-CL. Performed the experiments and analyzed the data: X-HN, S-CL, Z-KJ. Interpretation of the findings: X-HN, S-CL, Z-KJ and J-JY. All authors contributed to the article and approved the submitted version.This work was supported by the Postdoctoral Research Grant in Henan Province (grant number 1901004), the Henan Science and Technology Research Program (grant number 2018020142), and The Natural Science Foundation of Henan Province (212300410265). The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.https://www.cancer.gov/tcga), ICGC database (https://dcc.icgc.org/), and CPTAC (https://proteomic.datacommons.cancer.gov/pdc/study/PDC000127).The results shown here are in whole or part, based upon data generated by the TCGA Research Network (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background and Aims: Heart rate variability (HRV), i.e., the beat-by-beat fluctuations in heart rate (HR) reflecting the autonomic nervous system balance, is altered in patients with diabetes. This has been associated with arterial aging (stiffer arteries) and differs in men and women. The present study hypothesized that the impact of HRV on arterial aging, indexed as carotid–femoral pulse wave velocity (PWV), differs in a gender-specific manner and is affected by diabetes mellitus. Method: A total of 422 outpatients (187 women and 235 men) were studied. PWV was measured using the validated SphygmoCor device . Time-domain and frequency-domain parameters were measured to assess HRV. Results: The prevalence of diabetes was 30.8% with a slight, but nonsignificant, greater prevalence in men. Both age and SBP were independent determinants of PWV in each of the four groups (men and women with or without diabetes). Low-frequency activity was inversely correlated with PWV. It was greater in women without diabetes, but it was not significant in men regardless of the presence of diabetes. Conclusions: Beyond age, blood pressure, and diabetes, impaired cardiac autonomic function assessed by determination of HRV was significantly associated with arterial aging. The association between lower sympathetic and parasympathetic activity and stiffer arteries was significant in women, but not in men. An aging population is accompanied by emerging cognitive impairment  and a drThe prevalence of type 2 diabetes mellitus is increasing worldwide and significantly impacts on CV mortality . PreviouTo better characterize factors contributing to the modulation of arterial stiffness, we studied heart rate variability (HRV), i.e., the beat-by-beat fluctuations in heart rate (HR). HRV reflects the autonomic nervous system balance on the heart and is an indicator of CV risk  and mortAlterations in HRV are common in diabetes  and haveThe aim of the present study was to characterize gender-differences with regard to the impact of HRV on arterial stiffness and whether they differ according to the presence of diabetes.The study population consisted of subjects who visited our Outpatient Clinic for evaluation of their blood pressure levels. Therefore, the study population resulted in a combination of subjects who resulted hypertensive and/or were undergoing a periodic follow-up of their condition and of subjects who were “unaffected”/healthy, often undergoing the visit as requested by their fitness center. Patients were excluded if they had cancer, acute myocardial infarction in the previous 6 months, hepatic or cardiac failure, serum creatinine ≥2 mg/dL, secondary hypertension, or thyroid disease. Additional exclusion criteria were a previous large vessel stroke and atrial fibrillation, since its presence interferes with the accuracy of aorta stiffness measurement.After informed consent was given, all patients underwent a medical history, a clinic visit, urinalysis, electrocardiography, and traditional CV risk factor measurements.We analyzed 422 patients presenting complete measurements of all the parameters investigated. All the measurements were collected at the same visit. These patients will represent the study population of the current study.2.Hypertension was defined as the intake of antihypertensive medications and/or if the average of three blood pressure (BP) measurements taken at 5 min interval was ≥140 for SBP or ≥90 mm Hg for DBP. Diabetes was defined as the previous diagnosis and/or use of antidiabetic medications. Body mass index (BMI) was calculated as body weight (kg)/height (m)Aorta stiffness was assessed non-invasively by the carotid–femoral PWV. PWV was measured using the validated SphygmoCor device , whose validation and reproducibility have been previously published . Pulse tHRV was measured with the AtCor Medical HRV software to assess sympathetic/parasympathetic autonomic function. A standard 2 min electrocardiogram recording was performed with the patient in the supine position, with a regular and calm breathing pattern (5 min resting study) in a quiet room.Time domain measures are the mean and standard deviations of RR intervals recorded by the continuous electrocardiogram, where NN intervals represent all the RR intervals. The time domain of HRV was evaluated using the root mean square of successive differences between normal heartbeats (RMSSD) (m/s), which provides an estimate of the short-term components of HRV; the lower this vagal index, the greater the CV burden and the percentage of adjacent NN intervals that differ from each other by more than 50 m (pNN50) (%), which reflects alterations in autonomic function that are primarily vagally mediated and that are virtually independent of circadian rhythms.−power spectral density at the high-frequency (HF) range (0.15–0.4 Hz);−power spectral density at the low-frequency (LF) range (0.04–0.15 Hz);−the LF/HF ratio.The frequency domain of HRV consists of the spectral analysis of a series of consecutive RR intervals in order to quantify sympathetic and vagal influences on the heart. The evaluated parameters were:Vagal activity is the major contributor to the HF component and to the time-domain parameters, while LF reflects both sympathetic and vagal activity; the ratio LF/HF is considered to mirror sympathovagal balance .All analyses were performed using SAS University Edition.PROC UNIVARIATE was adopted to test for normality. Both the Kolmogorov–Smirnov test and Shapiro–Wilk test revealed that the measured variables were not normally distributed; therefore, differences between men and women were compared by the Wilcoxon rank sum test or Mann–Whitney U test (PROC NPAIR1WAY wilcoxon).Multiple linear regression models were constructed to identify the association of HRV with PWV—separately in men and women, with and without diabetes—independent of age, hypertension, and BMI. SBP, DBP, HR, RMSDD, LF, HF, and LF/HF were all introduced as covariates. Backward elimination was adopted to achieve a more parsimonious model.An ANCOVA analysis was employed to test for interaction between sex and diabetes.p value < 0.05 indicated statistical significance.A two-sided The study population consisted of 422 patients (187 women and 235 men). The prevalence of diabetes was 30.8% with a slight, but nonsignificant, greater prevalence in men.As illustrated in Multiple linear regression models were constructed to identify the significant determinants of arterial stiffness, indexed as PWV, according to sex and diabetic status. Regression model were adjusted for age, hypertension, and BMI. SBP, DBP, HR, RMSDD, LF, HF, and LF/HF were all introduced as covariates. Backward elimination of nonsignificant variables was adopted to achieve a more parsimonious model.The regression coefficient (with standard error) between the statistically significant factors in multivariable regression models and PWV are illustrated in To identify whether the impact of LF on PWV differed in men and women with or without diabetes, an ANCOVA analysis was conducted with interaction terms: sex × LF (does the correlation between LF and PWV differ in men and women?); diabetes × LF (does the correlation between LF and PWV differ in diabetic and nondiabetic patients?); sex × diabetes × LF (does the correlation between LF and PWV differ in men and women depending on their diabetic status?) .In the present study, we showed that beyond age, blood pressure, and diabetes, impaired cardiac autonomic function assessed by determination of HRV was significantly associated with abnormal PWV. The association between lower sympathetic and parasympathetic activity and stiffer arteries was affected by the presence of diabetes, and it was significant in women, but not in men.High sympathetic activity has been associated with stiffer arteries in patients with and without diabetes . This haA previous study reported that in diabetic patients, the presence of cardiac autonomic neuropathy was associated with reduced aortic distensibility , an assoFactors modulating and/or impacting on HRV, arterial stiffness, and their relationship are complex. They include smoking, dyslipidemia, level of physical activity, comorbidities, medication for diabetes, and coexisting disease. The TODAY Study reported that glycemic control in younger diabetic subjects was associated with greater sympathetic activity (LF/HF) and stiffer arteries . AdditioGender differences have been reported for several conditions affecting arterial stiffness and/or the autonomic control of the heart: BP ,22 and hConcerning the observed gender differences in the association between HRV, namely lower low-frequency activity and PWV, it has been previously reported that, despite greater resting HR in women than in men, heart rate does not have the same predictive power for CV mortality and morbidity in women as it does in men ,29. TherCharacterization of mechanisms underlying the observed gender differences in the association between the low-frequency domain of HRV and arterial stiffness is beyond the design of the present study. A better characterization of the underlying mechanism may improve management and therapeutic indications and pharmacological treatment to reduce the CV burden in diabetes.The present study has some limitations. The first is the cross-sectional design that does not allow determination of a causal relationship between PWV and cardiac autonomic. Another limitation is that we did not adequately characterize metabolic control in diabetic patients nor the full spectrum of drugs taken by the recruited patients. Given the high percentage of patients with hypertension, several medications  could interfere with HRV and with its association with arterial stiffness.To summarize, the results of the present study suggest that the impact of HRV, i.e., the differences in the autonomic regulation of mean HR, on PWV, a parameter that reflects arterial aging, differs in men and women with or without diabetes. Research on HRV needs to emphasize and report sex differences in healthy as well as in population affected by CV conditions.


The appropriate management of patients with Dravet Syndrome (DS) is challenging, given the severity of symptoms and the burden of the disease for patients and caregivers. This study aimed to identify, through a qualitative methodology and a Delphi consensus-driven process, a set of recommendations for the management of DS to guide clinicians in the assessment of the clinical condition and quality of life (QoL) of DS patients, with a special focus on patient- and caregiver-reported outcomes (PROs).This study was conducted in five phases, led by a multidisciplinary scientific committee (SC) including pediatric neurologists, epileptologists, a neuropsychologist, an epilepsy nurse, and members of DS patient advocates. In phases 1 and 2, a questionnaire related to patients' QoL was prepared and answered by caregivers and the SC. In phase 3, the SC generated, based on these answers and on a focus group discussion, a 70-item Delphi questionnaire, covering six topic categories on a nine-point Likert scale. In phase 4, 32 panelists, from different Spanish institutions and with a multidisciplinary background, answered the questionnaire. Consensus was obtained and defined as strong or moderate if ≥80% and 67–79% of panelists, respectively, rated the statement with ≥7. Phase 5 consisted of the preparation of the manuscript.The panelists agreed on a total of 69 items (98.6%), 54 (77.14%), and 15 (21.43%) with strong and moderate consensus, respectively. The experts' recommendations included the need for frequent assessment of patient and caregivers QoL parameters. The experts agreed that QoL should be assessed through specific questionnaires covering different domains. Likewise, the results showed consensus regarding the regular evaluation of several clinical parameters related to neurodevelopment, attention, behavior, other comorbidities, and sudden unexpected death in epilepsy (SUDEP). A consensus was also reached on the instruments, specific parameters, and caregivers' education in the routine clinical management of patients with DS.This consensus resulted in a set of recommendations for the assessment of clinical and QoL parameters, including PROs, related to the general evaluation of QoL, neurodevelopment, attention, behavior, other comorbidities affecting QoL, SUDEP, and QoL of caregivers/relatives and patients with DS. SCN1A gene  and the genetic syndrome with febrile seizures (FS or FS+) (Dravet syndrome (DS) is a life-threatening epilepsy syndrome that begins in infancy or early childhood and includes a wide spectrum of symptoms ranging from mild to severe . AdditioN1A gene , which e or FS+) –8.Dravet syndrome, first described in 1978 , althougSeveral guidelines and recommendations on DS are available, including a recently published European treatment guideline . HoweverThe Delphi technique is a structured method broadly used to collect relevant information on a specific issue and consists of a series of questionnaires or “rounds” targeted to experts . The keyad hoc questionnaire was designed for this project to assess the views of the patients and the scientific committee regarding aspects related to QoL, the impact of comorbidities on QoL, and caregivers' QoL. The coordinator of the project (Dr. Aledo) was in charge of designing the questionnaire. In the beginning, the QoL-related publications found by the literature search were examined. Relying on literature and clinical experience, the questions were developed , one neuropsychologist, one epilepsy nurse, one neuroscientist specialized in DEEs and working with patient organizations, and two members of DS patient advocates.The scientific committee selected a group of 32 epilepsy experts with a multidisciplinary background as participants . The criAn item or statement was considered consensual when there was “agreement” of opinion in the panel. This happens when the panelists' votes outside one of the three-point regions  containing the median were less than one-third of the answers (<33.3%). The median value determines the group consensus: majority “disagreement” if the median was within 1–3 and majority “agreement” if the median was within 7–9. Cases where the median was within the 4–6 region were considered “doubtful”. Conversely, “discordance” was considered when the scores of one-third or more of the panelists were in the –9] contaSPSS Statistics version 20 by IBM  was used to create and analyze the database. The median and the percentage of responses in the 7–9 range were calculated, and their values were used to define consensus.This study was performed following the Helsinki Declaration. Data from the Delphi questionnaire were anonymized for the analysis. All the personal data were dissociated from the results in compliance with the EU General Data Protection Regulation (GDPR).The 70 statements developed by the scientific committee covered a total of six categories: (1) general aspects of the QoL evaluation of patients with DS (15 items), (2) evaluation of neurodevelopment (24 items), (3) assessment of attention and behavior (9 items), (4) evaluation of other comorbidities affecting QoL (14 items), (5) Sudden unexpected death in epilepsy (SUDEP) (4 items), and (6) assessment of the QoL of caregivers/relatives (3 items). Of the 32 DS experts invited to participate in the Delphi process, a total of 28 (87.5%) answered the questionnaire . The panA 100% consensus was obtained across category 1 with an agreement on all the 15 statements, although 4 (26.7%) were categorized into the moderate consensus range . The panRegarding the content of the QoL questionnaires used to monitor patients, the experts reached a consensus that these tools should be specific to the syndrome and should consider the biological or chronological age of the patients. The questionnaires should include aspects related to the patient's physical health, the psychological status, the social environment and functioning, the economic situation, housing conditions and supporting material, adverse effects of pharmacological treatment, and the relationships of the patient and caregivers with healthcare professionals and the healthcare system.To conclude this category, the panelists reached a consensus agreement regarding the administration and assessment of the QoL questionnaire. The experts stated that an electronic format is an optimal method and that the questionnaire should be administered and evaluated by healthcare professionals, always in communication and coordination with each other and with the patient's family and caregivers.The panelists reached a consensus agreement on all 24 statements (100%) of category 2, of which 6 (25.0%) had a moderate consensus . In theiThe panelists agreed on the importance of assessing several comorbidities or complications related to cognitive impairment. Likewise, experts agreed on recommendations related to the evaluation and study of gait and ataxia, including the frequency of evaluation.Experts agreed on the likely suitability of a series of scales to measure neurological and motor development in patients with DS, including Bayley-III Cognitive Scale, Wechsler Intelligence Scale for Children-V, Peabody 2 Developmental Motor Scale-2, Behavior Rating Scale of Executive Function-2 (BRIEF-2), Gross Motor Function Classification System (GMFCS), Gillette Functional Assessment Questionnaire, and Pediatric Evaluation of Disability Inventory Computer Adaptive Test (PEDI-CAT). These scales should be used considering the specific objectives proposed by the adult/pediatric neurologist in collaboration with the neuropsychologist. Finally, the experts agreed on other parameters to be considered when assessing motor development, as well as on the frequency of these measurements.All the initial nine statements proposed in category 3 reached consensus agreement status , althougThe panelists overall agreed on category 4 items, obtaining consensus agreement on 13 of the 14 statements (92.86%), as shown in Overall, the panelists agreed on various recommendations related to the assessment of sleep-related problems , with the exception of the actigraphy study, for which consensus was undetermined. Additionally, the experts recommended spending time specifically explaining to the family and/or caregivers the potential comorbidities associated with seizures during sleep.Experts also recommended assessing other comorbidities at least every 3–6 months, including digestive and respiratory disorders, and available specific guidelines for vaccination periods and infectious processes for both family members and the physicians of the Healthcare Center.Consensus was reached on all 4 items in category 5 (100%), with a strong consensus for all of them . The panThe results of the category regarding the assessment of the QoL of caregivers/relatives are shown in To our knowledge, this is the first study to establish recommendations regarding QoL and PROs assessment and the main comorbidities in DS, using a Delphi technique and qualitative methodology, including caregivers and a multidisciplinary group of experts. Caregivers with a variety of familiar relationships and providing care to patients with a wide range of ages were included, and experts represented highly qualified professionals involved in the treatment and care of DS patients in Spain. The experts reached a consensus on recommendations for the management and follow-up of DS, focusing on the assessment of patients' and caregivers' QoL and other outcomes. The high degree of consensus reached by the panelists is noteworthy, with consensus agreement reached for 69 of the total 70 items proposed , coverinAppropriate assessment of clinical and QoL parameters of patients with DS is crucial due to the importance of the symptoms and burden of the disease , 30, 31.The recommendations regarding general aspects of QoL evaluation of patients with DS indicate that experts overall consider the evaluation of the patient's QoL at each visit with the clinician to be of great importance. This opinion is supported by several publications indicating an association between clinical parameters and the QoL status of patients with DS , 30, 34.For neurodevelopmental evaluation in patients with DS, the panelists recommended that assessments be conducted at each visit with the specialist, with varying frequencies depending on the patient's age. Given the neurodevelopmental delay in DS children –40, the Regarding the assessment of attention and behavior in patients with DS, the panelists recommended evaluating the behavior of patients with DS in the educational/stimulation center at each consultation with the specialist, with a frequency of at least 3–6 months. For this statement, the percentage of consensus reached 67.9%, close to the limit of agreement, which may be due to the indicated follow-up time. Several panelists commented that a follow-up of 6 months is sufficient and more in line with the reality of clinical practice, with limited time for each patient visit. The motivation for this recommendation is that behavioral problems in DS are very common and have a tremendous impact on these patients' lives . PatientDuring the assessment of other comorbidities affecting QoL in patients with DS, a total of 13 statements of the 14 proposed obtained a consensus agreement. The only item not validated in the entire Delphi questionnaire was the one related to the use of actigraphy study for evaluating a sleep disorder. The reason for this may be the scarcity of studies on this subject conducted in the SD population . The remSUDEP is the leading reported cause of death reported in DS, accounting for nearly half of all premature deaths . The expDS is a disease with a significant impact on patients' caregivers and relatives , 54. ForThe Delphi technique is widely used in health studies as a method to obtain useful information from experts when the published body of evidence is incomplete , 28, 55.The high burden on QoL for DS patients and their caregivers, as well as the lack of concise guidelines on the evaluation of various clinical parameters, makes it necessary to issue expert recommendations to fill these gaps. The present study intended to meet these needs through an expert consensus, which may be a useful guide for clinicians involved in the routine follow-up and management of patients with DS. In particular, this study focused on PROs, which constitute a patient-centered alternative to incorporate into future clinical practice of patients with DS, as classical measures fail to cover all aspects of the disease . FurtherThe 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 from the patients/participants or patients/participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.Adrián García-Ron, Alberto Vieco, Alfonso Amado Puentes, Helena Alarcón Martínez, Irene García Morales, Javier Aparicio Calvo, Jesús Eirís Puñal, Juan Jesús Rodriguez Uranga, Juan José Poza Aldea, Julián Lara Herguedas, Julio Ramos-Lizana, Mercè Falip Centelles, Mercedes Garcés Sánchez, Miquel Raspall Chaure, Patricia Smeyers Dura, Rafael Toledano Delgado, Rocío Calvo Medina, Salvador Ibáñez Micó, Sergio Aguilera Albesa, Víctor Soto Insuga, and Xiana Rodríguez Osorio.ÁA-S has contributed to the design and conception of the study. All authors have contributed to the acquisition, analysis, interpretation of the data, have participated in the drafting and reviewing of the manuscript, and approving the submitted version.This study was funded and promoted by Zogenix Spain, now a part of UCB.Author ÁA-S has received funding for educational and research activities from Angelini, Zogenix, UCB, PTC pharma, Blueprint genetics, GW, Eisai. Author AG-N has received advisory or research funds by Arvelle/Angelini, Bial, Biocodex, EISAI, Esteve, GW Pharma, Jazz Pharmaceuticals, PTC Therapeutics, Stoke, UCB Pharma and Zogenix. Author JA is president of the Dravet Syndrome Foundation Spain (DSF). He and/or the DSF have received grants and/or financial support from GW Pharma, Zogenix, Ovid Therapeutics, Encoded Therapeutics, Biocodex, Praxis, Health in Code, Takeda, UCB, Epygenix, Jazz Pharmaceuticals and StrideBio to help carry out some of the DSF's foundational activities or provide consulting services. Author JA honoraria have always been donated directly or indirectly to the DSF. Author RS-C has been a trial investigator for GW Pharmaceuticals, Takeda Pharmaceutical Company Ltd. Author and Zogenix and has served on advisory boards for Novartis, GW Pharmaceuticals, Biocodex and Zogenix. Author JG-P has received advisory or lecture funds by BIAL, EISAI, GW, NUTRICIA, SANOFI, UCB and ZOGENIX. VV has participated in advisory boards and symposia organized by Angellini, Bial, Eisai Inc., GW pharma, Novartis, Takeda, UCB, Zogenix. Author AM was employed by Dracaena Consulting SL. Author LA was employed by Apoyo Dravet. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.