An increased prevalence of adolescent metabolic syndrome (MS) is associated with adulthood cardiovascular diseases. This study aimed to explore the potential relationship of quality of life (QoL) and personality traits with adolescent MS.A total of 1961 participants from Chongqing with an average age of 11.68 years old from a cohort study established in 2014 and followed up through 2019 were included. QoL information, Eysenck’s personality questionnaire and MS components were collected.P < 0.01), which was mainly negatively correlated with the MS components of central obesity, diastolic blood pressure (DBP) and triglyceride levels, as well as positively correlated with high density lipoprotein cholesterol (HDL-C) level. The total QoL score was negatively correlated with triglyceride levels and positively correlated with DBP . High extraversion personality score was a protective factor against adolescent MS (P = 0.04) and MS score (P < 0.05), which were mainly negatively correlated with the MS components of waist circumference, systolic blood pressure and TGs, and positively correlated with HDL-C .A higher QoL domain score of physical activity ability (PAA) was a protective factor for both MS and MS score (all QoL score and extraversion personality score were independent protective factors against both MS prevalence and MS score, suggesting that community intervention to improve the QoL and psychological health of children are essential.The online version contains supplementary material available at 10.1186/s12955-021-01797-7. The increased prevalence of adolescent metabolic syndrome (MS) and MS component severity scores are independent predictors of adulthood cardiovascular diseases (CVDs) , 2. MS cLimited studies  have illPersonality traits can impact MS and its components, although few studies have investigated this association. One study showed that children with obesity might experience several psychosocial problems . A studyIn this study, we investigated whether QoL scores were inversely correlated with adolescent MS and MS components, which domains have salient effects, and whether the association was independent of age, sex, region and other variables. We investigated whether personality traits  were associated with MS and MS components, and whether the association was independent of the influence of sex, age and other factors. A hypothesized framework figure is drawn in Additional file Two stage stratified cluster sampling was used to include participants from two counties in Chongqing that represent urban and rural areas; then, two regions per county were randomly selected, and 3067 children  were ultimately informed and included if they met the inclusion criteria. Participants who met all of the following criteria were recruited: (1) aged between 6 and 9 years old in 2014; (2) resided in the target region for more than 6 months; (3) did not have serious diseases ; and (4) had consent provided by the parents and children for participation. The sample size was calculated with the following parameters: α level of 0.05, power of 90%, prevalence of MS components of 15% and prevalence in the population of 10%, using the formula Demographic information, SES and prenatal variables were collected. The validity and reliability of the demographic questionnaire were checked and are described in detail in a previous publication . The demAnthropometric measurements were conducted both in 2014 and in 2019 by well-trained paediatric nurses, and the protocol for these measurements was described in a previous publication . Waist cBlood pressure (BP) was measured on three separate occasions with an OMRON arm-type electronic sphygmomanometer (HEM7051) using an appropriately sized BP cuff placed on the subject’s right arm and with the subject in a seated position, as described in detail in a previous publication .The biochemical markers FBG, HDL-C, TG, low density lipoprotein cholesterol (LDL), and total cholesterol (TC) were measured in 2014 and 2019. Venous blood (3 mL) was drawn from each subject in the morning after at least 12 h of fasting and 24 h of abstaining from high-fat and spicy foods. The biochemical markers were measured within 2 h after venous blood was drawn using the protocol introduced in detail in our previous publication .The QoL questionnaire for adolescents consists of 49 items, including 4 factors  and 13 dimensions, including self-satisfaction, relationship of teacher and pupil, physical feeling, companionship, parenthood, physical activity ability (PAA), learning ability and attitude, self-esteem, negative emotion, attitude towards completing homework, opportunity for activity, living convenience and others (picky-eating and surroundings), as detailed in Additional file The Chinese version of the Eysenck personality questionnaire consisteFifty samples were required to complete the same questionnaire twice within a one-week interval to check the validity and reliability of the QoL and Eysenck’s Personality Questionnaires before our formal survey. QoL and Eysenck’s Personality Questionnaires were completed by the adolescents after a standard training.MS in adolescents was defined by the presence of three or more of the following five components , 25: (1)The definitions of size for gestational age were based on the global reference for foetal-weight and birthweight percentiles : birth wP < 0.05), and compared the differences among MS component groups . The χ2 test was used to test the difference in the component ratio of potential risk factors for MS components. A logistic regression model was performed using diagnosed MS components or MS as the dependent variables with QoL and personality traits, as independent variables, and adjusted for covariables. In addition, a generalized linear model (GLM) was used to analyse the correlation of QoL and personality trait scores with MS component levels and MS scores and adjusted covariables.Differences in anthropometric measures, serum biochemical indexes, QoL and personality score among the three groups were assessed using ANOVA, and post hoc comparisons were performed using the Student–Newman–Keuls (SNK) test. Type I errors were already adjusted. We selected potential covariates if the difference was significant . A significant difference was defined by an α level of 0.05.P < 0.05).The general characteristics of the subjects are shown in Table P < 0.01, P = 0.03 and P < 0.01). In addition, the extraversion score of personality traits was decreased in children with MS compared with their counterparts with no MS components or 1–2 MS components (P = 0.05).Table P < 0.01) and living environment factor scores (OR (95% CI) 0.948 , P < 0.01) were protective factors against central obesity. However, the domain score of others (picky-eating and surroundings) was positively correlated with central obesity (OR (95% CI) 1.014 , P = 0.03), elevated BP (OR (95% CI) 1.027 , P < 0.01) and decreased HDL (OR (95% CI) 1.024 , P = 0.01). The QoL domain scores of self-satisfaction, physical feeling, negative emotion and factor scores of physical and mental health and quality of life satisfaction were positively correlated with elevated BP (in Table P = 0.02) were negatively correlated with elevated BP. In addition, an elevated domain scores of QoL of self-satisfaction, physical feeling, parenthood, PAA, negative emotion, factor score of physical and mental health and total score of QoL were correlated with elevated TGs, while neuroticism score was positively correlated with elevated TGs (in Table P = 0.04) and activity opportunity (OR (95% CI) 1.054 , P = 0.03) were positively correlated with IFG.The association of elevated MS components in 2019 with adolescent QoL and personality traits in 2019 is shown in Additional file P < 0.01) and living environment (OR (95% CI) 0.934 , P = 0.006) were negatively correlated with central obesity, companionship was positively correlated with elevated BP (OR (95%CI) 1.037 , P = 0.04), and activity opportunity was positively correlated with IFG (OR (95%CI) 1.102 , P = 0.02). Furthermore, extraversion personality was a protective factor against elevated BP (OR (95% CI) 0.974 , P = 0.03).Subgroup analyses of participants without MS components in 2014 are shown in Table P < 0.01). In addition, a high extraversion personality score was a protective factor for MS even after adjusting for sex, age, region, foetal weight of pregnancy week and mother’s education level (OR (95% CI) 0.984 , P = 0.05). However, the QoL score of the “other” domain (picky eating and surroundings) was positively correlated with MS (OR (95% CI) 1.029 , P = 0.01). In the full model, the QoL domain of others (picky-eating and surroundings) was positively correlated with MS (OR (95% CI) 1.048 , P < 0.01), and the living environment factor was negatively correlated with MS (OR (95% CI) 0.932 , P < 0.05).In the multivariable logistic regression model Table  (adjusteP < 0.01). In contrast, a high QoL score of the “other” domain was positively correlated with the MS score . In the full model, other (picky-eating and surroundings) domains were positively correlated with MS even after adjusting for covariates , and extraversion personality was a protective factor against MS in both the total sample  and subgroup  analyses after adjustment for covariates.After adjusting for other covariates, the GLM Table  also revThis study is the first prospective cohort study revealing both the longitudinal and cross-sectional correlations between QoL and MS or MS components, as well as the association between personality traits and MS or MS components over an average 12-year follow-up from birth to adolescence in urban–rural areas. We observed that an increased prevalence of MS components and MS, as well as MS score, was associated with QoL score and extraversion personality traits. The QoL domain score of PAA and extraversion personality score were independent protective factors for both MS and MS scores.Our study revealed that centrally obese children and adolescents were mainly negatively correlated with physical QoL scores, such as PAA and factor score of living environment, and a dose-dependent relationship was found. Our result was consistent with that of other studies –33, showResearch on the relationship between glycolipid indexes and QoL is limited. A study of adult cardiovascular disease illustrated that elevated TG levels were inversely associated with both physical and QoL scores . Our stuHowever, a cross-sectional study showed that QoL scores were correlated with an increase in the components of MS, and the physical health domain of QoL had the most significant association . In our Personality traits can be associated with MS, but the conclusion remains controversial, and few studies have been conducted in adult populations; no related cohort studies have explored this relationship in adolescents. The results from Japanese adults  showed tOur study has some limitations that should be considered when interpreting the results. First, since this study was a bidirectional cohort study, recall bias could exist for the prenatal variables. We checked the birth certificates to verify the birth weight, stature and gestational age. Second, data on GH and gestational diabetes were collected through a questionnaire, and recall bias existed. However, we collected the same perinatal information in both 2014 and 2019 independently to reduce recall bias and nonresponse. Finally, QoL and personality traits were collected in a cross-sectional manner in 2019, making it difficult to draw conclusions regarding the causality relationships of QoL and personality traits with MS.In conclusion, the prevalence of MS was elevated among adolescents. Our findings add to the scarce research evidence regarding the relationships of QoL and personality traits with MS in children and adolescents, which could facilitate the generation of policy suggestions for health management institutes. In this study, we identified the relationships of QoL and personality traits with MS and MS components from both prospective and cross-sectional aspects after adjustment for other covariates. First, the PAA score was inversely correlated with MS and MS scores, suggesting that the establishment of a comprehensive community, family and school intervention model to improve the PAA of children is essential for the control of adolescents with MS. Second, the QoL score of other domains, including picky eating and surroundings, was positively correlated with MS and MS scores, indicating that health education, including literacy training and health dietary habits, should be implemented both at home and in school to improve lifestyles. Finally, the extraversion personality trait was a protective factor against MS and MS score; therefore, primary prevention in childhood health should focus on cultivating children's extraversion personality. Further studies should explore the effect of comprehensive intervention on the control of adolescent MS by improving QoL and personality traits.Additional file 1: Figure S1. The hypothesized framework of this manuscript.Additional file 2: Table S1. Comparation baseline characteristics between included and excluded participantsAdditional file 3: Figure S2. The structure chart of quality of lifeAdditional file 4: Table S2. Logistic regression analysis of the association of QoL and personality traits with adolescent MS components without adjusting other covariates


There is increasing concern regarding cardiovascular risk in non-alcoholic fatty liver disease (NAFLD) patients with liver fibrosis. This study aims: (1) to assess the association between NAFLD and liver fibrosis status and the development of carotid plaque (CP), and (2) to identify CP risk factors among general population with different baseline NAFLD and liver fibrosis status.This retrospective cohort study included 14,288 adult participants who went for regular health check-ups between 2014 and 2019, in one hospital in Zhejiang, China. NAFLD was diagnosed by abdominal ultrasound and the NAFLD fibrosis score (NFS) was calculated to reflect the extent of liver fibrosis. Cox proportional hazards analyses were applied to assess the risk of CP development across groups with different baseline NAFLD and NFS status.p < .001). Progression from NAFLD free and NAFLD with low NFS to NAFLD with high NFS are associated with 1.56-fold  and 1.43-fold  increased risk of CP, respectively. Risk factors associated with CP vary based on baseline NAFLD and NFS status. Among NAFLD participants with high NFS, hypertension is the only significant risk factor after adjustment for other potential influencing factors.NAFLD participants with high NFS had higher risk of CP compared to non-NAFLD participants  and liver fibrosis status can be an independent predictor for development of carotid plaque.Progression from NAFLD free and NAFLD with low NAFLD fibrosis score (NFS) to NAFLD with high NFS are associated with increased risk of carotid plaque.Risk factors associated with carotid plaque vary based on baseline NAFLD and NFS status, and hypertension plays the most important role among patients with NAFLD and high NFS.NAFLD and liver fibrosis status can be an independent predictor for CP development regardless of metabolic abnormalities. Hypertension is a major risk factor for CP development among NAFLD patients with high NFS. Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the world, and its prevalence is up to 25.24% in the general population and over 76% in type 2 diabetics (T2DM) ,2. NotabCarotid plaque (CP), a lipid-driven inflammatory vascular disease, is a surrogate for subclinical CVD and is a predictor of cardiovascular events ,8. AccumWe hypothesised that NAFLD and liver fibrosis status are associated with the development of CP in the general population. To test this hypothesis, we performed a large retrospective cohort study among individuals who went for regular health check-ups. Based on health check-up results data, we examined factors that potentially contribute to the development of CP among people with different NAFLD and liver fibrosis status.This retrospective cohort study included individuals who went for regular health check-ups between January 2014 and December 2019, in the Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang, China. Inclusion criteria were as follows: age ≥ 18 years; had both abdominal and carotid ultrasound check-ups between 2014, January 1 and 2014, December 31 and were CP free; had at least one follow-up check-up including both abdominal and carotid ultrasound before 2019, December 31; the baseline and follow-up check-ups had to be at least 6 months apart. Subjects with any of the following conditions were excluded from this study: alcohol drinking history; hepatitis B virus positive; chronic liver diseases. The flow diagram for subject selection process is presented in Medical history of hypertension, DM, chronic liver diseases, personal history of drinking and smoking were systematically acquired by physicians. Blood pressure and anthropometric were measured as described in our previous study . Venous Carotid and abdominal ultrasound examinations were routinely performed in a health check-up at our health management centre by experienced ultrasonographers following a standardised protocol. High-resolution sonography machines  were used for carotid ultrasound with a 10-MHz probe and for abdominal ultrasound with a 3.5-MHz probe. CIMT was measured the vertical distance from the upper edge of the intima to the upper edge of the adventitia on the far wall of the distal 10 mm of the common carotid artery. CP was defined as an IMT >1.5 mm in any portion of the carotid arteries . Fatty l2) + 1.13 * impaired FPG/DM  + 0.99 * AST/ALT ratio − 0.013 * platelet count (× 109/l) − 0.66 * albumin (g/dl)  vs Q1 [≤5.2%], HR 1.54, 95% CI 1.28–1.86, p < .001; Q3 [5.5–5.6%] vs Q1 [≤5.2%], HR 2.07, 95% CI 1.71–2.50, p < .001; Q4 [≥5.7%] vs Q1[≤5.2%], HR 3.30, 95% CI 2.76–3.96, p < .001) , Cr  and sUA  were associated with increased risk of CP.For people who are NAFLD free at baseline, hypertension  increased risk of CP; however, those with obesity only but no MetS had 0.26-fold decreased risk of CP .For the group with NAFLD and low NFS at baseline, participants with hypertension, who are smokers and with elevated HbA1c levels have increased risk of CP. Cr and gender were also included in the model based on stepwise model selection results; however, the association between the two factors and CP risk were not significant after adjusting for other selected variables. Obesity and MetS were also found to be predictors of CP. Further analysis indicated that the interaction effect between the two factors was significantly associated with the development of CP. Among people with NAFLD and low NFS, compared to people with neither obesity nor MetS, those with MetS but no obesity had 1.41-fold . Cr level may be a potential influencing factor; however, the risk of CP over different Cr quartile levels did not differ significantly after adjusting for hypertension status.Among people who already had NAFLD with high NFS, the risk of CP development was significantly associated with hypertension  with sufficient follow-up period (5 years). On the other hand, our study was performed in general population which are representative for healthy population without specific health concern.In summary, our findings suggest that NAFLD and liver fibrosis status can be an independent predictor for CP even after adjusting for gender and metabolic abnormalities. Progression of NAFLD and fibrosis status during follow-up may increase the risk of CP. Risk factors for CP varied by baseline NAFLD and fibrosis status, and hypertension plays the most important role for development of CP among subjects with NAFLD and high NFS. The CP risk factors we identified among people with different NAFLD and liver fibrosis status were completely based on regular health check-up results, indicating that effective evaluation of risk of CP development during regular health check-ups are clinically practicable. Moreover, tailored intervention strategies for reducing CP risk could be developed given patients’ baseline NAFLD and liver fibrosis status and to target on specific risk factors varied by the baseline status.


Study Design. This study is a randomized clinical trial (RCT). Participants. Twenty-three nonvitrectomized pseudophakic eyes of 23 subjects (9 M and 14 F with mean age: 54.8 years) with refractory DME due to ERM.  To compare the efficacy and safety of suprachoroidal and intravitreal injection of triamcinolone acetonide in pseudophakic patients with refractory diabetic macular edema (DME) due to epiretinal membrane (ERM). n = 13 eyes) or intravitreal triamcinolone acetonide 4 mg/0.1 ml (IVTA) (n = 10 eyes) and were evaluated at baseline and 1 and 3 months after injection to assess outcome measures. Main Outcome Measures. Changes in best-corrected visual acuity (BCVA) (primary outcome), central foveal thickness (CFT) by optical coherence tomography (OCT), and intraocular pressure (IOP) measurement (secondary).  The eyes were randomized to suprachoroidal triamcinolone acetonide injection 4 mg/0.1 ml (SCTA) , and 1st (P=0.605) and 3rd months (P=0.313). Regarding mean CFT, no significant differences were observed at baseline (P=0.353) and at the first month (P=0.214) between both groups, while at the third month, CFT was significantly higher in the IVTA group (385 um) than in the SCTA group (323 um) (P=0.028). Mean IOP was significantly higher in the IVTA group (15 mmHg) on 1st month than in the SCTA group (12 mmHg) (P=0.011); after 3rd month, IOP was significantly higher within the IVTA group (18 mmHg) than SCTA (14 mmHg) (P=0.028). No significant difference was noted between both groups at baseline IOP (P=0.435).  Baseline median BCVA (logMAR) was 1.0 (range 0.8–1.0) in both groups, improved within the SCTA group to 0.8 on the 1 Both SCTA and IVTA are effective in reduction of CFT and improvement of patients' visual acuity, but with a higher recurrence rate and rise in IOP after IVTA when compared to SCTA. Both treatments have temporary effects with the possibility of recurrence of DME and the need for retreatment. DME can occur at any stage of diabetic retinopathy (DR), although it is more likely to occur as the disease progresses . Poor glThe incidence of epiretinal membrane (ERM) formation in DME increases with various risk factors, such as old age, cataract surgery, and panretinal photocoagulation . PreviouCorticosteroids have both anti-inflammatory and angiostatic effects, they have been used in the form of intravitreal injections or implants as a second-line therapy in the treatment of DME due to their side effects as cataract progression and ocular hypertension, in refractory DME due to ERM corticosteroids therapy is considered the first line of treatment due to their effect on various inflammatory mediators .Dexamethasone implant (DI) is a common form of steroids used in cases of refractory DME due to ERM; a recent study conducted by Erden et al. concluded that the presence of ERM limited efficacy of DI in reducing CFT and the recurrence of DME occurred at the same baseline levels after 6 months of follow-up with a significant increase in intraocular pressure (IOP) , 11, 12.Suprachoroidal injection is a novel therapy for intraocular drug delivery with higher concentration towards posterior segment of the eye compared to intravitreal route . Hulk trIn this study, our objective was to compare efficacy and safety of suprachoroidal and intravitreal injection of triamcinolone acetonide in the pseudophakic eyes with refractory diabetic macular edema due to epiretinal membrane with no vitreomacular traction.In this randomized clinical trial, twenty-three pseudophakic eyes diagnosed as refractory DME due to ERM were enrolled in the retina clinic at Ophthalmology Department, Benha University Hospital, Egypt, and Ebsar Eye Center, Benha, Egypt, from February 2020 to April 2021.The findings of the ophthalmological examination were included. Best-corrected visual acuity (BCVA) was measured with the Snellen chart and then converted to logMAR. IOP was measured by Goldmann applanation tonometry . The diagnosis of diabetic retinopathy and macular edema was made with slit-lamp using auxiliary +90D lens and fundus fluorescein angiography, central foveal thickness, and presence of ERM by OCT .The patients were randomly assigned to one of two groups. The suprachoroidal triamcinolone acetonide group (SCTA) group (number of eyes: 13) received a single injection with a dose of 4 mg/0.1 ml and the intravitreal triamcinolone acetonide (IVTA) group (number of eyes: 10) received a single injection with a dose of 4 mg/0.1 ml. Patients were followed one and three months after injection to assess changes in BCVA, CFT, and IOP from baseline measures.Written informed consent was obtained from all patients included in this study after discussing the surgical procedures to be performed and possible side effects of it.This study was approved by the Faculty of Medicine, Benha University, and was carried out in compliance with principles of the Declaration of Helsinki.µm detected by OCT with posterior vitreous detachment and no anteroposterior vitreomacular traction, pseudophakic nonvitrectomized eyes with refractory DME due to ERM, and had no treatment for DME in the last three months at the time of recruitment. Our exclusion criteria were as follows: preexisting retinal disease other than diabetic retinopathy that may affect final visual outcome, ischemic diabetic macular edema detected by fundus fluorescein angiography, epiretinal proliferation that causes lamellar macular hole or contracted ERM detected by OCT, the glaucomatous eyes in which IOP ≥21 mmHg measured by Goldmann applanation tonometry and/or asymmetric vertical cup disc ratio or glaucoma suspect patients, central corneal opacity, vitreous hemorrhage, uncooperative patients or patients with poor fixation, and patients on systemic medications that could affect the macular thickness and uncontrolled systemic diseases.Our inclusion criteria were as follows: type II diabetes mellitus, centrally involving DME with CFT greater than 300 Surgical techniques were performed in the operating room under complete sterile conditions by the same surgeon. The surgical preparations were the same for both injection techniques, topical anesthetic eye drop  was instilled into the conjunctival sac, 10% periocular povidone-iodine was applied on the skin, and 5% povidone-iodine was applied on the conjunctiva after insertion of the sterile speculum and eye draping. We used 4 mg/0.1 ml of triamcinolone acetonide  using the nonfiltering sedimentation technique for the purification of triamcinolone acetonide suspension.Suprachoroidal injection was done using custom made needle preparation to expose only 1 mm of the 27-gauge needle to enter the suprachoroidal space; we used the silicon tube with the sterile 22-gauge blue IV cannula  to use it as a guard for the 27-gauge needle ; after cutting the silicon tube from its base, we insert the 27-gauge needle inside; then, we cut the silicon tube by Westcott scissor to expose only 1 mm of the 27-gauge needle bevel using a sterile caliper to measure the desired length. Surgeon aspirates 0.1 ml of triamcinolone suspension into a sterile 27-gauge syringe and then inserts the custom 27-gauge suprachoroidal needle we prepared. Paracentesis was created; then, suprachoroidal injection was done 3.5 mm posterior to the limbus in the inferotemporal quadrant.Intravitreal injection was placed 3.5 mm posterior to the limbus after paracentesis using a standard 27-gauge needle (1 ml insulin syringe and 27 g needle).Fourth-generation fluoroquinolone eye drop is used 4 times per day for one week for all patients , and patients were instructed for follow-up schedule 2 days, one week, and one and three months after the procedure. BCVA, CFT, and IOP were measured for all patients one and three months after surgery for each group.t-test or Mann–Whitney U test for normally and nonnormally distributed numerical variables, respectively. Categorical data were compared using the chi-square test. Within-group comparisons were made using repeated-measures ANOA or Friendman's test for normally and nonnormally distributed numerical variables. All statistical tests were two-sided. P values less than 0.05 were considered significant. Kaplan–Meier was done to estimate time to recurrence in both groups.Data management and statistical analysis were done using SPSS version 25 . Quantitative data were assessed for normality using the Shapiro–Wilk test and direct data visualization methods. According to normality testing, numerical data were summarized as means and standard deviations or medians and ranges. Categorical data were summarized as numbers and percentages. Quantitative data were compared between study groups using the independent Twenty-three eyes of 23 patients were included in this study .P value = 0.808) and gender  (No significant differences were observed between the two groups regarding age (= 0.431) .P value = 0.927), 1st month , and 3rd month   .rd month . Post hoc analysis revealed that it was significantly higher at baseline (1 logMAR) than 1st and 3rd months (0.8 logMAR for each). Furthermore, the IVTA group showed a significant improvement in BCVA at 3rd month . Post hoc analysis showed that it was significantly higher at baseline (1.0 logMAR) than at the first and 3rd months   .P value was 0.028. No significant differences were noted at baseline  and at 1st month  between both groups.The central foveal thickness at three months was significantly higher in the IVTA group (385 um) than in the SCTA group (323 um); the P value <0.001). Post hoc analysis revealed that it was significantly higher at baseline (541 um) than at the 1st and 3rd months . Also, within the IVTA group, CFT showed an overall significant improvement . Post hoc analysis revealed that it was significantly higher at baseline (512 um) than at the 1st and 3rd months   .rd month CFT was 331 um, while in the SCTA group (d) baseline CFT was 658 um, (e) on 1st month, CFT was reduced to 275 um, and (f) on 3rd month CFT was 302 um.P value = 0.011. Furthermore, in the third month, it was significantly higher in IVTA (18 mmHg) than in the SCTA group (14 mmHg). P value was 0.028. No significant difference was noted between both groups at baseline   than in the SCTA group (12 mmHg). = 0.435) .P value = 0.02). At one month, the recurrence rate was 50% in the IVTA group and 0% in the SCTA group. After 3 months, the recurrence rate was 70% in the IVTA group and 30.8% in the SCTA group  and two eyes in the IVTA group (20%); we faced no cases with inadvertent intravitreal injection among SCTA; whereas, the Hulk trial reported one case of inadvertent Intravitreal injection .Both SCTA and IVTA have a short-term effect in improving BCVA in refractory DME due to ERM with the possibility of recurrence; however, SCTA has a lower incidence of ocular hypertension and recurrence of DME; this study has several limitations, such as relatively small sample size and short-term follow-up, and more studies are needed to investigate the long-term efficacy of SCTA in cases of DME with ERM and the possibility of repeated injections of SCTA injections to achieve a long-term anatomical and functional improvement with the delay of surgical removal of ERM in these cases.


Schistosomiasis is one of the most prevalent parasitic infections in the developing world. When it affects the gastrointestinal system specifically the liver, it causes periportal fibrosis followed by cirrhosis. Cholecystitis however is a rare presentation, and associated liver abscess has certainly never been reported to date. We report a case of acute cholecystitis complicated by cholangitis and liver abscess in a 46-year-old man. After complex course of treatment, he had laparoscopic cholecystectomy, and the histology report confirmed schistosomiasis. Gallbladder schistosomiasis is an uncommon disease that is associated with dense fibrotic changes that strongly mimics xanthogranulomatous cholecystitis. Liver abscess may occur during the disease evolution especially in patient originating from endemic backgrounds. We present the case and a comprehensive literature review. Parasitic infections remain a problem in the developing countries . Schistomansoni, schistosomiasis of the gallbladder (GB) is remarkably uncommon [In spite the fact of the high frequency of hepatic involvement particularly by Schistosoma uncommon . About tuncommon .A 46-year-old Egyptian man presented to the emergency department with one-day history of epigastric and right upper abdominal pain, associated with nausea and vomiting. He denied any other associated gastrointestinal or urologic symptoms. Apart from being type 1 diabetic, he declared no other significant past medical history.The patient presented in good shape, and there was no hemodynamic instability. He showed epigastric tenderness with no signs of peritonism with the rest of the abdomen unremarkable. White blood cell count (WBC) was 17.4 K/uL (4-10 K/uL). Liver function tests (LFTs) were abnormal: total bilirubin 47 umol/L (0-21 umol/L), direct bilirubin 35 umol/L (0-3 umol/L), ALT 156 U/L (0-40 U/L), and AST 182 U/L (0-41 U/L). Other results showed hemoglobin 14.4 g/dL (13-17 g/dL), platelets 190 K/uL (150-400 K/uL), lipase 106 U/L (13-60 U/L), CRP < 5 mg/L (0-5 mg/L), CA 19-9 303 U/ml (0-27 U/ml), and IgE 432 Ku/L (0-114 Ku/L). IgA and IgG4 were normal. Abdominal ultrasound (US) showed multiple GB stones with intrahepatic biliary dilatation and prominent common bile duct (CBD) measuring 7 mm. The rest of the examination showed no signs of acute cholecystitis and no bile duct stones. The patient was admitted as a case of obstructive jaundice for further work up. He later developed signs of sepsis (tachycardia and fever) for which blood cultures were taken. He was started on intravenous ceftriaxone and metronidazole. Endoscopic retrograde cholangiopancreatography (ERCP) showed purulent bile immediately following cannulation and failed to show any filling defects. Sphincterotomy and CBD stenting were done. ERCP procedure was not extraordinary in difficulty to suspect ampullary fibrosis or deformation. Magnetic Resonance Cholangiopancreatography (MRCP) later showed distended GB containing sludge and tiny stones, hyperenhancement of both GB and CBD walls, and mildly thickened GB wall in addition to pericholecystic edema and fat stranding, consistent with acute cholecystitis and cholangitis. There were no CBD stones nor thickening of the CBD wall. After stenting, the patient was kept on piperacillin/tazobactam. He improved clinically, and both his LFTs and inflammatory markers were trending down till the 4th day post-ERCP when he started to spike fever again. Septic work up was repeated, and endoscopic ultrasound (EUS) was done. This exam showed the stent in place. Both US and computed tomography (CT) showed a new lesion 5 × 4 × 5 cm in the segment IVb of the liver in continuation with the GB fundus . The lesDuring follow-up visits, the patient was asymptomatic. ERCP was done 5 weeks after discharge and showed no filling defects in the CBD. The stent was removed. The patient travelled and was lost to follow-up for 5 months. When he came back, an MRCP showed complete resolution of the liver abscess. Multiple gall stones in GB were still demonstrated. He was booked for elective laparoscopic cholecystectomy. Intraoperative findings showed omental adhesions to the GB. The GB was also densely adherent to the liver. Despite the difficult dissection, the procedure was managed laparoscopically. Patient's postoperative course was unremarkable, and he was discharged next day after surgery. Histopathology of the gallbladder showed chronic cholecystitis, with granulomatous inflammation secondary to schistosomiasis .The first case of gallbladder schistosomiasis (GBS) was reported in 1975, and since then, speculations were made regarding possible pathogenesis . FourteeThe risk factor for contracting schistosomal infection is the contact of its larval form with the skin through contaminated water in endemic areas . Most ofClinical presentation is variable according to the involved organ. Infestation of urinary tract may lead to hematuria, fibrosis, and obstructive uropathy that may lead to parenchymal renal damage . When itCholangitis is mostly caused by ascending bacterial infection due to obstruction . Causes Liver abscess has already been described as a complication of liver schistosomiasis . HoweverThe imaging modality of choice for gallbladder disease is abdominal ultrasound, but it has no specific signs to indicate GBS . In our For GBS, the treatment remains surgical, usually by laparoscopic cholecystectomy . At surgMoreover, four patients received postoperative complementary medical treatment which was praziquantel , 15, 19.The specimen pathology usually reveals a lymphocytic infiltrate; schistosomal eggs can be found in any layer of the gallbladder wall causing fibrocalcific reaction; most of the cases showed granulomatous inflammation surrounding the schistosomal eggs .GBS might be considered preoperatively in patients who lived in an endemic area and developed symptoms suggestive of gallbladder disease. This is the first case that report a liver abscess in a patient with cholecystitis with a gallbladder infested by Schistosoma. However, a majority of cholecystitis in patients with schistosomiasis involve the presence of gallstones. This condition carries the same possible complications and should be managed in the same way as usual cholecystitis. Surgeons must however expect a more difficult dissection during operation.


Garugapinnata Roxb. (Burseraceae) is a medium-sized tree widely available all over the tropical regions of Asia. Bryophyllum pinnatum (Lam) Oken. (Crassulaceae) is an indigenous and exotic plant grown in tropical regions. Both plants have been used for their anti-inflammatory, antioxidant, anticancer, wound healing, antidiabetic activities, etc. This investigation was designed to explore the result shown by methanolic extract of Garuga pinnata bark and Bryophyllum pinnatum leaves, on cognitive power and retention of the memory in experimental mice along with quantification of phenolic compounds and DPPH radicals neutralizing capacity. The memory-enhancing activity was determined by the elevated plus-maze method in Scopolamine-induced amnesic mice, using Piracetam as allopathic and Shankhpushpi as ayurvedic standard drugs. Two doses (200 and 400 mg/kg p.o.) of both extracts were administered to mice up to 8 consecutive days; transfer latency of individual group was recorded after 45 minutes and memory of the experienced things was examined after 1 day. DPPH assay method and the Folin–Ciocalteu method were employed to determine antioxidant potency and total phenol amount, respectively. 400 mg/kg of the methanolic B. pinnatum bark extract significantly improved memory and learning of mice with transfer latency (TL) of 32.75 s, which is comparable to that of standard Piracetam (21.78 s) and Shankhpushpi (27.83 s). Greater phenolic content was quantified in B. pinnatum bark extract (156.80 ± 0.33 µg GAE/mg dry extract) as well as the antioxidant potency . Our study proclaimed the scientific evidence for the memory-boosting effect of both plants. Memory is a vital process by which experiences are recorded and can be used to adapt their responses to the environment. Alzheimer's disease (AD) is a major condition related to memory impairment and it can be characterized as a continuous neurodegenerative disorder that can lead to senile dementia, particularly in the cortex and hippocampus region. Besides distinct clinical manifestations, behavioral hallmarks involve progressive loss in memory, judgment capacity impairment, decision-making problem, physical atmosphere orientation, and language problem . StudiesConvolvulus pluricaulis Choisy are approved for medicinal uses [Clitoria ternatea Linn., Canscora decussata Schult., and Evolvulus alsinoides Linn. are also categorized as Shankhpushpi by Indian practitioners [Shankhpushpi is a widely used herbal drug in the management of cognitive disorders, from the times of Acharya Charak (200 BC) to date . Shankhpnal uses . Based oitioners . It is mitioners , 6. Shanitioners . Neutralitioners , 9.Bryophyllum pinnatum (Crassulaceae) is abundantly distributed in the tropical and subtropical regions around the world. Aerial parts or entire plant can be utilized to manage hypertension, syphilis, jaundice, candidiasis, dysmenorrhea, convulsions, and so forth. Many bioactive compounds, like bryotoxin A, B, C, caffeic acid, and protocatechuic acid, have been reported from this plant [is plant  that exhis plant ; antiurois plant .Garuga pinnata (Burseraceae) is a deciduous plant found in India and east and south parts of Asia. It has been reported that G. pinnata extracts exhibited antiulcer, anticancer, antibacterial, antidiabetic, and antioxidant activity [B. pinnatum and bark of G. pinnata is used by the ethnic groups of the Tharu community to enhance memory. However, there is no scientific evidence on this ethnomedicinal claim. Thus, our study is mainly focused on investigating the memory-improving effect of these two plants along with their antioxidant activity.activity . EthnomeThe chemicals used are Piracetam and Scopolamine hydrobromide , Shankhpushpi , ascorbic acid (Merck), DPPH (Sigma-Aldrich), sodium bicarbonate (Fisher Scientific), hydrochloric acid, mercuric chloride, potassium chloride, ferric chloride, lead acetate, and Kagnesium ribbon (SD fine-chem limited).G. pinnata and B. pinnatum were collected from Boadgoau, Kapilvastu, Nepal  during September 2019 and were identified by National Herbarium and Plant Laboratories, Nepal. The herbarium voucher specimens of the plants have been deposited in the pharmacognosy laboratory of Crimson College of Technology (CCT-HRB-075-187 (G. pinnata) and CCT-HRB-075-188 (B. pinnatum)). Bark and leaves of both plants were shade dried; comminuted to fine powder and extraction was carried out by the triple maceration process using methanol as the menstruum. The extracts were dried with the help of the rotary vacuum evaporator at 45°C and sticky dried extract was obtained which was further stored at 4°C until use.Preliminary screening for the presence of secondary metabolites in the sample was performed by using previously described methods with slight modifications –16.μg gallic acid equivalent (GAE)/mg extracts.For the determination of TPC, the Folin–Ciocalteu method was adopted . TPC wasThe potency of the plant extracts to neutralize the DPPH free radicals was determined by the previously established method . EthanolA0 denoted the DPPH solution absorbance and A1 denoted the test sample absorbance.Here, Male and female Swiss albino mice (25–30 g) were placed inside polypropylene cages with standard feed and water ad libitum. Animal handling, care, and experimental design were conducted according to the guidelines of the Institutional Review Committee, Nepal Health Research Council (Ref. no. 622/2019). Experimental animals were acclimatized for two weeks before experimental procedures . GuideliElevated plus-maze and Scopolamine-induced amnesia were used for the exteroceptive and interoceptive behavior design, respectively, to monitor the cognitive power and retention of the memory, according to a previously established method with slight modification .The elevated plus-maze having 2 open arms (16 × 5 centimeters), 2 closed arms (16 × 5 × 12 centimeters) raised vertically with the 25-centimeter height was applied. Individual experimental animals were kept at the edge of the open arm facing far from the middle rostrum; then, total duration for moving from the edge of the open arm to both closed arms using all legs  was noted. Those animals that failed to reach inside any closed arm in a period of 90 sec were kindly propelled toward closed arms and TL was considered to be 90 s. The mice were permitted to search the maze for the next 10 s and afterward come back to the cage. Memory of this experienced work was investigated 1 day after the initial examination day.L0 is the transfer latency after 1 day and L1 is the first transfer latency expressed as seconds.Transfer latency after 1 day was interpreted in the terms of Inflection Ratio (IR), by applying the following equation:n = 6). All groups except for vehicle control (Group I) were treated with Scopolamine hydrobromide (0.4 mg/kg) intraperitoneally, on the eighth day of extract/standard drug treatment to induce amnesia. Transfer latency was recorded after 45 minutes and the memory of experienced things was evaluated after 1 day in every classification:Group I : vehicle controlGroup II : Scopolamine controlB. pinnatum leaves test sample (200 mg/kg)Group III : B. pinnatum leaves test sample (400 mg/kg)Group IV : G. pinnata bark test sample (200 mg/kg)Group V : G. pinnata bark test sample (400 mg/kg)Group VI : Group VII : Shankhpushpi (5-fold dilution)Group VIII : Piracetam (100 mg/kg)A total of 48 mice were classified into 8 groups  and considered significant if G. pinnata bark and B. pinnatum leaves was 12.04 and 10.46%, respectively. Qualitative phytochemical examination revealed the occurrence of phenolic compounds, flavonoids, tannins, saponin, and alkaloids. The entire phenolic amount was quantified by utilizing the calibration curve of the standard gallic acid  (EC50 50.14 μg/mL) and methanolic extract of B. pinnatum leaf (MPBL) extract (EC50 109.41 μg/mL) revealed that they have a moderate antioxidant capacity in comparison to ascorbic acid standard (EC50 5.47 μg/mL). Furthermore, the activity depends upon the dose used. The free radical neutralizing capacity of MGBP is greater as compared to the MBPL.According to After the continuous monitoring of MBPL and MGPB oral ingestion at different doses , it was concluded that plant extracts have no acute toxicity.th and 9th day whereas, at the higher dose (400 mg/kg), it exhibited an incredible reduction in TL , on the 8th and 9th day, suggesting noteworthy enhancement in cognitive and remembrance power (p < 0.005) on the 8th and 9th day. Scopolamine hydrobromide  injected on the 8th and 9th days has high TL, indicating retardation in cognitive and remembrance power. Oral administration of the MBPL and MGPB at optimum doses (200 and 400 mg/kg up to 8 regular days) effectively improved Scopolamine produced memory loss. The results obtained from the plant extracts are comparable to the standard Piracetam and Shankhpushpi. However, Shankhpushpi showed better results than all the plant extracts.We verified the memory-enhancing activity of the two ethnomedicinally claimed plants for the first time. Oral ingestion of the MBPL (200 mg/kg) for 8 consecutive days did not produce any prominent result on TL, on the 8ce power . MGDP exG. pinnata (156.80 ± 0.33 µg GAE/mg) than in B. pinnatum leaves (27.782 ± 0.25 µg GAE/mg) reported in our study correlates the greater antioxidant activity observed and corresponds to the previous findings [Preliminary screening of phytochemicals is an important step for detecting various bioactive secondary metabolites of plants, having a vital role towards beneficial medicinal and physiological activities such as antioxidant, antidiabetic, and anticancer activities . Our stufindings . Plant-dfindings .G. pinnata bark is more effective in inhibiting DPPH free radical compared to methanolic extract of B. pinnatum in all the tested concentrations. The higher amount of phenolic compounds might be the reason for better antioxidant potency of the G. pinnata bark extract. In a previous study, 40% of DPPH free radicle was found to be inhibited by 100 µg/mL of methanolic B. pinnatum extract [DPPH free radical inhibition percentage data from  extract . In anot extract .G. pinnata and B. pinnatum might be a major contributing factor for observed memory-enhancing activity [G. pinnata may have contributed to its higher memory-enhancing effect as compared to B. pinnatum in this study. In a previous study on the Swiss albino mice model, an aqueous extract of B. pinnatum (AEBP) at 200 mg/kg body weight had significantly controlled the sleeping time and other overall behaviors, induced by phenobarbitone along with delaying effect on the onset of picrotoxin and strychnine induced convulsion. B. pinnatum extract might exert depressive effects on the central nervous system as a result of its GABAergic and less commonly glycinergic transmission [B. pinnatum was also supported by a previous study, in which AEBP leaves had shown improvement of short-term memory loss by suppressing CCL4 induced acetylcholinesterase activity. The leaf extract was very much prominent to reduce CCL4 induced nitric oxide (NO) and malondialdehyde (MDA) overproduction in the brain to protect from oxidative stress. Increased MDA level is an indication of oxidative stress to result in oxidative damage of the brain. Oversynthesis of NO diminishes the function of antioxidant enzymes, hence leading to neurodegenerative diseases such as AD and depression [G. pinnata contain different types of diarylheptanoids [B. pinnatum and bark of G. pinnata were reported to present potent anti-inflammatory nonsteroidal derivatives [β-amyrin structure, and so on [G. pinnata bark and B. pinnatum leaves as a medicinal plant in Nepal.An elevated plus-maze model was adopted to determine the memory-enhancing efficacy of plant extracts in which Scopolamine was injected to induce amnesia in mice . Howeveractivity , 35. Oxyactivity . Also, tactivity . Consequsmission . Memory-pression . Acetylcpression . Acetylcpression . Adminispression , 39. Theptanoids , 42, whiptanoids , 44. Besivatives , phytostnd so on , 46. Fland so on , 48. Simnd so on . The preG. pinnata and B. pinnatum both contain bioactive phytoconstituents like alkaloids tannins, phenols, and flavonoids with free radical scavenging and memory-boosting ability. So, it is possible to utilize these plants for the management of cognitive impairment, related to AD. Besides, the significant memory-enhancing activity of these plants may be associated with their phenolic content and antioxidant potency. Further study is needed at the preclinical and clinical level with the elucidation of the exact mechanism.


This study identified two infant AD case definitions that were strongly associated with known AD risk factors. These case definitions can be used to study novel AD risk factors in large cohort studies, potentially providing new insights into the epidemiology of infant AD. Enrollment was conducted at 17 hospitals across 14 US states during 2011 to 2014 winter seasons Data .8 The 2.2At enrollment, investigators conducted a structured interview with parents/guardians to assess patients' demographic characteristics, family and medical history, and clinical details; blood specimens were collected. After the bronchiolitis hospitalization, study staff interviewed parents/guardians by telephone at 6‐month intervals, in addition to medical record review by trained physicians.2.3The exposures were known predictors of infant AD, collected by parent report at enrollment: maternal history of AD, paternal history of AD, either parent with history of AD, maternal history of allergic rhinitis, and maternal history of asthma.The outcome measures were a panel of case definitions for infant AD. During both enrollment and the age 12‐month interview, parents/guardians were asked if the infant had a history of eczema/AD, defined as an “itchy, scaly rash that comes and goes.”We compiled the following case definitions: (a) parent‐reported AD from interviews; (b) clinician‐diagnosed AD from medical records; (c) hybrid approach ; (d) hybrid plus elevated tIgE; (e) hybrid plus elevated sIgE; and (f) hybrid plus elevated tIgE or sIgE.2.4Analyses were performed using Stata 14.2 . We calculated descriptive statistics using percentages and medians with interquartile ranges (IQR), overall and by AD definition. We assessed associations of AD risk factors with AD case definitions using log‐binomial models to calculate risk ratios (RRs) and 95% confidence intervals (95% CIs). Regression models used a clustered sandwich estimator to account for potential clustering by site. Complete case analysis was used for missing data.3The study enrolled 1016 infants, of whom 921 (91%) were followed longitudinally ; 95 (9%) contributed enrollment data only . We excluded two participants who died during infancy, one from the analytic and one from the nonanalytic cohort. The analytic and nonanalytic cohorts did not significantly differ in demographics Table .In the analytic cohort, the median age was 3 months (IQR 2‐6), 60% were male, and 43% were non‐Hispanic white. AD prevalence ranged from 55/919 (6%) for hybrid plus elevated sIgE to 268/920 (29%) for parent‐reported AD Table . Among 2In regression models, the strongest associations of AD risk factors were observed with parent‐reported infant AD Table . For exa4The prevalence of infant AD ranged from 6% to 29% across definitions, encompassing AD prevalence estimates from previous cohort studies of healthy Polish (17%)Although clinical examination by a dermatologist remains the gold standard for AD diagnosis,Dharma et al, however, reported that questionnaire data cannot accurately substitute for assessment by healthcare professionals using validated criteria in diagnosing infant AD.This study has limitations, most notably the lack of validation against clinical examination by a dermatologist. Moreover, our sample consisted of infants with severe bronchiolitis. Although bronchiolitis is the most common reason for hospitalization of US infants,5We compared potential case definitions for infant AD and propose the use of: (a) parent‐reported AD and (b) hybrid approach (parent‐reported and clinician‐diagnosed AD) plus elevated tIgE or sIgE. These results can inform future epidemiologic studies of novel risk factors for infant AD and the atopic march.Mr. Zheng was supported by the Case Western Reserve University School of Medicine Dean's Award . This study was supported by the grants U01 AI‐087881, R01 AI‐114552, and UG3 OD‐023253 from the National Institutes of Health . The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The supporting sources had no involvement in the study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication.The authors report no conflicts of interest relevant to this work.Conceptualization: David X. Zheng, Ruth J. Geller, Carlos A. Camargo Jr.Data curation: Ruth J. Geller.Formal analysis: Ruth J. Geller.Funding Acquisition: Carlos A. Camargo Jr.Investigation: Lacey B. Robinson, Markus D. Boos.Methodology: David X. Zheng, Ruth J. Geller, Carlos A. Camargo Jr.Project administration: Markus D. Boos.Supervision: Carlos A. Camargo Jr.Validation: Lacey B. Robinson.Writing–Original Draft: David X. Zheng.Writing–Review and Editing: Ruth J. Geller, Lacey B. Robinson, Markus D. Boos, Carlos A. Camargo Jr.All authors have read and approved the final version of the manuscript.Carlos A. Camargo Jr. had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.David X. Zheng affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.Data S1. Supporting Information.Click here for additional data file.Figure S1. Overlap between three definitions of infant atopic dermatitisThe three definitions shown are parent‐reported atopic dermatitis, clinician‐diagnosed atopic dermatitis ascertained from medical record review and physician‐ascertained atopic dermatitis based on parent report and medical record review (“hybrid” definition).Click here for additional data file.Figure S2. Overlap between elevated total IgE (tIgE) and specific IgE (sIgE) among infants with atopic dermatitis (“hybrid” definition)Elevated sIgE includes positive results to any food allergen using ImmunoCAP or positive results to any food or aeroallergen using Immuno Solid‐phase Allergen Chip. “Hybrid” atopic dermatitis refers to physician‐ascertained atopic dermatitis based on parent report and medical record review. IgE, immunoglobulin EClick here for additional data file.


Amending from ancient classic, Ziyin Tongluo Formula (ZYTLF) has been prescribed to treat postmenopausal osteoporosis (PMOP) for decades with good curative effect. However, the possible mechanisms of it are still unknown.Ovariectomized rat model was established to validate the therapeutic effect of ZYTLF on PMOP by Micro-CT bone analysis and pathological observation. Subsequently, active ingredients of ZYTLF and corresponding putative targets were identified by online databases. Overlapping genes were first obtained from mining genes associated with PMOP and then overlapped them with the putative targets. Key genes were selected from the multiple constructed and analyzed networks. GO and KEGG pathway enrichment analysis were performed by importing the key genes to the DAVID database. Moreover, validation of the binding association between key targets and their corresponding active compounds were accomplished by AutoDock Tools and other software. Lastly, Enzyme linked immunosorbent assay (Elisa) detection and Western blot analysis were utilized to further explore the possible mechanism of ZYTLF on PMOP.With 129 target genes interacting with PMOP, 92 active compounds of ZYTLF corresponded to 243 targets, and 50 key genes were chosen. Network analysis revealed the top 10 active ingredients, such as quercetin and kaempferol and the top 50 key genes, such as ERα, p38 MAPK, p-AKT and TGF-β1. Enrichment analysis uncovered multiple signaling pathways, including estrogen signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. Furthermore, our finding of the foremost active compounds was tightly bound to the core proteins, which were verified by molecular docking analysis. Through experimental studies, we confirmed that the prescription of ZYTLF could ameliorate the OVX-induced bone loss, suppress the osteoclast activity and boost osteoblast ability through experimental studies.The potential mechanisms and therapeutic effects of ZYTLF against PMOP may be ascribed to inhibition of osteoclast activity, boost of osteoblast activity and enhancement of the expression of ERα.The online version contains supplementary material available at 10.1186/s13020-021-00503-5. Postmenopausal osteoporosis (PMOP) is regarded as a severe chronic metabolic bone disease. There is a growing number of postmenopausal women around the world suffering from increased fragility and fracture susceptibility because of decreased ovarian function and estrogen level every year. At this stage of life, the bone resorption is faster than the bone building. Therefore, the incidence rate of osteoporosis steeps up in the population of women at menopausal age with plummeted estrogen level. Factors such as age, gender, genetics, reproductive status, calcium: phosphorus ratio, and exercise have certain impact on bone strength. Bone strength is consisted of bone mineral density (BMD) and bone quality. BMD is detected with dual energy absorptiometry (DXA) and declined by 30–40% at the age of 70s. However, there are many elderly patients who present with vertebral fractures but have bone density within the normal range in the ward. Bone quality deterioration is also closely associated with fracture , 2. WithHormone replacement therapy (HRT) in postmenopausal women enhances their estrogen levels and effectively reduces bone resorption, but its safety is still controversial . A meta-Radix Rehmanniae Praeparatae (Shu Di) as the monarch herb, Ophiopogon japonicus (Mai Dong), Fructus Ligustri Lucidi (Nv Zhen Zi), Radix Angelicae Sinensis (Dang Gui), Radix Paeoniae Alba (Bai Shao) as the minister herb, Loranthus parasiticus (Sang Ji Sheng), Achyranthes bidentata (Niu Xi), Caulis Spatholobi (Ji Xue Teng), Zaocys (Wu Shao She), Scolopendra, Radix Astragali (Huang Qi), Saposhnikoviae Radix (Fang Feng), Rhizoma Atractylodis Macrocephalae (Bai Zhu) as the assistant herb, Radix Glycyrrhizae Preparata (Gan Cao) as the guide herb. Radix Rehmanniae Praeparatae and its derivatives have been reported exerting bone protecting effect in the osteoporosis model by maintaining homeostasis between osteoclastogenesis and osteoblastogenesis [Ophiopogonin D, which was isolated from the Ophiopogon japonicus, showed anti-osteoporosis effect via reducing reactive oxygen species in vivo [Fructus Ligustri Lucidi is proved to have the function of improving bone metabolism and bone quality in ovariectomized, growing, aged and diabetic rats through the regulation of OPG/RANKL/cathepsin K and other signaling pathways [With specific therapeutic activity and low side effect, ZYTLF has been prescribed for decades in Guangdong Province Hospital of Traditional Chinese Medicine to prevent and treat postmenopausal osteoporosis. Our previous study demonstrated that ZYTLF could effectively alleviate clinical symptoms in patients and increase their bone density by inhibiting bone resorption, reducing bone turnover and improving bone microstructure . As a foogenesis . Ophiopo in vivo . Meanwhipathways . In addipathways . The menNetwork pharmacology is an emerging technology, which integrates systems biology and bioinformatics and other emerging interdisciplinary disciplines to reveal the relationship between drugs and the body from the perspective of biological systems network. Li first applied network pharmacology to TCM and proposed the concepts of “TCM network pharmacology” and “network target” , 17. In In this work, network pharmacology was applied to mine the key ingredients, targets and signaling pathways of ZYTLF against PMOP. Moreover, molecular docking simulation was utilized to validate the stability of key proteins and corresponding compounds by detecting binding affinity. Furthermore, ovariectomized (OVX) rat models was established to confirmed the therapeutic effect of ZYTLF on PMOP and elucidate its preliminary mechanisms Fig. .Fig. 1ScThe name and content of fourteen herbs in ZYTLF are shown in Table Experimental animal ethics panel of Guangdong Provincial Hospital of Traditional Chinese Medicine (License No. 2019044) approved the animal experiments. Experiments were performed in China Academy of Chinese Medical Sciences Guangdong Branch (License No. 00228297). Sixty three-month old female Sprague-Dawly (SD) rats, weighting 200–230 g, were purchased from the Laboratory Animal Center of Southern Medical University (License No. 44002100022874) and were allowed to acclimatize for one week before free access to food and space. All animal procedures complied with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. We tried our best to attenuate the number and suffering of the animals.The rats received either sham operation  or bilateral ovariectomy . All operations were performed with the application of anesthesia methods and sterile techniques as well as postoperative anti-infection and analgesia. Three months after ovariectomy, if the BMD of the rats were decreased significantly, the establishment of OVX animal model wound be considered to be successful.Five groups of qualified rats were randomly divided ). The rats in the SHAM group and OVX group were given distilled water by gavage, the OVX + ZYTLF Low/Middle/High dose group were administrated 1.34 g/kg, 2.68 g/kg, 4.02 g/kg per day respectively, and the OVX + WM group were treated with alendronate 10.51 mg/kg per week and calcitriol 0.075 μg/kg per day for 12 weeks. Rat intake dose was modified according to half, one or two times of the clinical human dose. The conversion obeys the Human-Rat Equivalent Dose Conversion Principle.After the last 24 h of medicinal treatment, overdose of anesthesia was used to euthanize the animals. The rats were anesthetized after three months. Blood samples from the abdominal aorta were first centrifuged at 4000 rpm for 10 min. Then the serum was removed and the samples were stored at – 20 °C. Lumber vertebra body and bilateral femur and tibia were also removed as samples.The contents of estradiol (E2), alkaline phosphatase (ALP), procollagen I N-terminal propeptide (PINP), tartrate-resistant acid phosphatase (TRACP), type I collagen cross-linked Amino Terminal Peptide (NTX), type I collagen cross-linked carboxyl Terminal Peptide (CTX-1), calcium (Ca), phosphorus (P) in rat serum samples were confirmed by commercial ELISA kits based on manufacturer’s instructions. The ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China.Bone structural and mineral changes in rats were evaluated by micro-computed tomography (micro-CT) technique. A micro-CT imaging system  was applied to perform the CT scan and trabecular morphometric analysis under the guidance of manufacturer’s application notes. A spatial resolution of 9 μm  was used to scan right femur samples. After that, CT images were reestablished by in-built software CT-vox and CTAn, respectively. With recommended order set, CTAn selected the trabecular region of interest in an unbiased batch. The aim of performing trabecular analysis was to quantify morphometric calculations and BMD.The measured direct trabecular metric parameters of right femur were as follows: trabecular thickness (Tb.Th), bone volumetric fraction (BV/TV), trabecular number (Tb.N) as well as trabecular separation (Tb.Sp). Calculations of directly measured non-metric parameters were performed as well, including the trabecular bone pattern factor (Tb.Pf), an inverse assessment of trabecular connectivity and the structural model index (SMI), an estimate of the prevalence of plate-like or rod-like trabecular.The left tibial tissue was acquired and fixed in 4% paraformaldehyde for 24 h and then decalcified for 2 weeks in 10% EDTA buffer (pH 7.0). The samples were dehydrated, embedded in paraffin and cut into slices (about 4 μm thick). Slices were processed with tartrate-resistant acid phosphatase (TRAP)-staining and alkaline phosphatase (ALP)-staining respectively, aiming to detect the number of osteoclast and osteoblast. The sections were visualized by using an optical microscope  and then photographed. The cytoplasm of osteoclasts was wine-red and the cytoplasm of osteoblasts showed grayish-black granules after ALP + TRACP staining.http://tcmspw.com/tcmsp.php) [http://bionet.ncpsb.org/batman-tcm/) [http://www.megabionet.org/tcmid/) [Radix Glycyrrhizae Preparata serves as the guide herb in ZYTLF with a small amount, greater bias was avoided by setting the standard as OB ≥ 60% and DL ≥ 0.36. The targets of ZYTLF were collected by TCMSP analysis platform and then were transferred to standard protein name in Uniprot database (https://www.Uniprot.org), setting organisms as Human [Fourteen herbs of ZYTLF were in inputted into the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP msp.php) . BAT-MANan-tcm/)  and Trad/tcmid/)  in sequeas Human .http://www.genecards.org) [http://omim.org/) [https://www.disgenet.org/) [“Postmenopausal” and “Osteoporosis” were the key words to gather PMOP connected genes from the three following online databases: Genecards (rds.org) , OMIM (him.org/) , and Diset.org/) . OverlapThe network model of “herb-compound-overlapping gene” was built by importing the overlapping target genes, their corresponding active components and herbs into Cytoscape 3.7.1 . CytoNCAhttp://string-db.org/, ver.11.0) [Construction of protein–protein interaction (PPI) network of overlapping proteins was completed through setting the condition of data analysis mode as “Multiple proteins”, the type as “Homo sapiens” (human), and the minimum mutual threshold as “high confidence (0.700)” in The STRING database (er.11.0) . The othhttps://david.ncifcrf.gov/) was utilized to conduct enrichment analysis of Gene Ontology  and Kyoto Encyclopedia of Genes and Genomes  [The Database Visualization and Integrated Discovery system  , 31, sethttps://pubchem.ncbi.nlm.nih.gov/). Procession and transformation of the 2D structure into PDB format were accomplished by PyMOL, and they were saved in PDBQT format as docking ligands. Concurrently, the collection of all the crystal structures of the key proteins from the RCSB protein data bank  and the selection of those with distinctive ligands and comparatively higher resolution were done. AutoDock Tools was used to take away the water molecules, isolate proteins and reserve them as receptors. The receptors and ligands were processed with PyMOL and Auto Dock and then docked through Vina.The binding efficiency of the overlapping proteins and major active components in ZYTLF were evaluated by computer simulation docking technology. The SDF structure of top 10 core compounds were collected in the network from the PubChem database (Radioimmunoprecipitation assay (RIPA) buffer that contained Halt Phosphatase Inhibitor Cocktail  were ground with right tibia samples, which were corrected based on the results of the bicinchoninic acid assay (BCA). 10% SDS-PAGE separated 30 µg protein. Then they were transferred to polyvinylidene fluoride (PVDF) membranes . In order to maximize the protein loading, membranes were air-dried and reactivated in methyl alcohol. According to the purpose of the experiment, they were blocked with 5% nonfat milk powder at room temperature, and incubated with primary antibody, including ERα , TGF-β1 , p38 MAPK , p-AKT , and GAPDH  at 4 °C overnight. The nitrocellulose membranes were washed three times with PBST and incubated with goat anti-rabbit IgG antibody  at room temperature for 2 h. After these procedures, they were rinsed three times and scanned for optimal density value of the target protein unit by improved Journal Pre-proof chemiluminescence analysis .All statistical analyses of this paper were performed by GraphPad Prism 8.0.2 . The data were represented by means ± standard deviation (SD). The nonparametric Kruskal–Wallis method was used for the data without Gaussian distribution based on Shapiro–Wilk test, followed by Dunn’s multiple comparisons test. One-way ANOVA analysis was used for the data with Gaussian distribution and equal SDs, followed by Dunnett’s multiple comparisons test. Brown-Forsythe ANOVA test was used for the data with Gaussian distribution and unequal SDs, followed by Dunnett’s T3 multiple comparisons test. Statistical analyses were conducted to confirm the differences between test groups and nominated control group, with the significance level at P < 0.05.To determine the threptic effects of ZYTLF on OVX rats, we collected serum from experimental rats after treatment. Related indicators in serum were detected by ELISA, which the result of were shown in Fig. To further determined the effect of ZYTLF on experimental animals, Micro-CT evaluation was performed on right femur. As shown in Fig. The micro-CT plain scan  of the distal femoral bone were shown in Fig. To verified the effect of ZYTLF on the activity of osteoclast and osteoblast, ALP + TRACP staining was conducted on bone biopsy. Figure A total of 1738 compounds were obtained from TSMSP platform, BAT-MAN TCM and TCMID database, among which 92 met the requirement were regarded as active components. Additional file A network of “herb-compound-overlapping gene” was established Fig. b. AdditiPerformance of GO enrichment and KEGG pathway enrichment analysis on 50 core genes and extraction of significant enrichment results (FDR < 0.05) were done by DAVID database. Totally, 62 GO-BP terms, 6 GO-CC terms, 11 GO-MF terms and 59 terms on the KEEG pathway were obtained. The enrichment results showed that the main biological processes were positive transcriptional regulation signals of RNA polymerase II promoter, negative regulation of apoptosis, positive regulation of transcription using DNA as a template, aging, positive regulation of gene expression, etc. Main cell components such as nucleus, cytoplasm and extracellular space were the major components of biological processes. Major regulated molecular functions were binding of enzyme, transcription factor and protein binding Fig. a–c. PMOPWhether the top 10 compounds were essential in regulating the 50 key proteins was verified by molecular docking simulation. A stable structure wound be formed when the ligand bound to one or more amino acid residues in the active site  of the receptor and took part in the process such as conformation change and energy complementation to combine with the receptor. This research illustrated that the top 10 compounds had sturdy association with 50 core proteins, such as AKT1 (PDB id: 6npz), MAPK14 (PDB id: 6sfo), ERα (PDB id: 4pxm), TNF (PDB id: 3m2w), TGF-β1 (PDB id: 6om2) and PTGS2 (PDB id: 5f19). These compounds were of great importance based on the result of network pharmacology , middle(p < 0.05) and high(p > 0.05) dose of ZYTLF. Moreover, ZYTLF showed similar effect to alendronate on trabecular microstructure indexes. The results indicated that ZYTLF could mitigate microstructure degeneration in OVX rats and preserve thicker, interwoven and plate-like trabecular bone. These observations demonstrated that ZYTLF could ameliorates trabecular bone resorption in OVX induced rat model with high turnover bone metabolism. Furthermore, our bone pathological analysis with ALP + TRACP staining and detection of bone metabolic markers confirmed that ZYTLF could ameliorate the bone phenotype of OVX rats, reduce the osteoclast formation and increase osteoblast differentiation. Many TCM therapy has an effect on promoting OB generation and inhibiting OC differentiation, as ZYTLF does. However, the specific mechanism of ZYTLF on osteoclastogenesis and osteoblastogenesis is still uncertain.Preliminary determination of the pharmacological compounds and complex molecular mechanisms of ZYTLF on PMOP were done by molecular docking technology and network pharmacology. Network pharmacology is a systematic approach employed to learn the complexities among compounds, targets, diseases and biosystems. It coincides with the holistic and systemic views of TCM theory . CurrentAdditional, network-based algorithm was utilized on key proteins to understand the gene ontology (GO) and KEGG pathways in the treatment of ZYTLF. From the result, we assumed that ZYTLF might manipulated multiple biological processes and molecular functions, consisted with the complexed mechanisms of PMOP. The data revealed 50 putative targets were involved in pharmacological action of ZYTLF on PMOP, in which ERα, p38 MAPK, p-AKT, TGF-β1 were considered as hub proteins. Moreover, 15 KEGG pathways were observably enriched by KEGG analysis, including estrogen signaling pathway, TNF signaling pathway, PI3K-AKT signaling pathway, MAPK signaling pathway, which were closely bound with the development and progression of PMOP. Estrogen bound with the estrogen receptor in osteoblasts and osteoclasts to act on the OPG/RANK/RANKL signaling pathway, which further propelled secretion of OPG, down-regulated the expression of RANKL, and inhibited the formation of osteoclasts . It is wIt is generally considered that estrogen deficiency is the major cause of the occurrence and development of PMOP, so the current research mainly focuses on how to elevate estrogen level. Interestingly, this research found that ZYTLF did not significantly boost estrogen level, but reduce bone loss to normal bone homeostasis. Through western-blot analysis, we found that ZYTLF treatment increased the expression of ERα protein in bone sample. ERα has been proved to play a critical role in bone metabolism. The expression of ERα in bone tissues is estrogen-dependent, and the transcription and translation of ERα are inhibited after the level of estrogen is declined . On the Compare our study with the Network pharmacology evaluation method guidance . There aIn summary, our findings illuminated that ZYTLF could ameliorate the OVX-induced bone loss, suppress the osteoclast activity and boost osteoblast ability. Moreover, the key compounds and target proteins were mined, such as quercetin, kaempferol and ERα, p38 MAPK, p-AKT and TGF-β1, which were considered as the materials of ZYTLF in treatment of PMOP. In addition, multiple signaling pathways, including estrogen, MAPK, PI3K/Akt and OPG/RANK/RANKL signaling pathways, involved the treatment mechanisms. Consequently, this study provides rational treatment of ZYTLF on postmenopausal osteoporosis with experimental evidence and preliminarily indicated its mechanism.Additional file 1: Table S1. The detailed information of active ingredients of ZYTLF.Additional file 2: Table S2. List of ZYTLF-targets, PMOP-genes, and ZYTLF-PMOP overlapping genes.Additional file 3: Table S3. Detail information of "Herb-Active ingredient-Overlapping gene" network.Additional file 4: Table S4. Results of Gene Ontology (GO) terms and pathways.Additional file 5: Table S5. The binding energies of ten core compounds and fifty key proteins.


In some breast cancer (BC) patients, an examination of lymph nodes dissected during sentinel lymph node biopsy (SLNB) demonstrates a presence of metastatic lesions and extracapsular extension (ECE) in a SLN. This study aimed to evaluate clinical relevance of ECE in BC patients. This is a retrospective analysis of 891 patients with cancer metastases to SLN, referred to supplementary axillary lymph node dissection (ALND), hospitalized between Jan 2007 and Dec 2017. Clinical and epidemiological data was evaluated. Long-term treatment outcomes were analysed. In 433 (48.6%) patients, cancer metastases were limited to the SLN (group I), in 61 (6.8%) patients the SLN capsule was exceeded focally (≤ 1 mm—group II). In 397 (44.6%) patients, a more extensive ECE was found (> 1 mm—group III). Metastases to non-sentinel lymph nodes (nSLNs) were diagnosed in 27.0% patients from group I, 44.3% patients from group II and in 49.6% patients from group III. No statistically significant differences were observed in long-term treatment outcomes for compared groups. The presence of ECE is accompanied by a higher stage of metastatic lesions in the lymphatic system. The differences in this respect were statistically significant, when compared to the group of ECE(−) patients. ECE, regardless of its extent, did not impact the long-term treatment results. ECE remains an indication for supplementary ALND and for other equivalent cancer treatment procedures, regardless of ECE size. An extracapsular extension (ECE) is defined as a presence of lymph node capsule perforation by metastatic lesions with accompanying infiltration of perinodal space5. When ECE is found in SLN, an ALND appears to be the method of choice.A diagnosis of an invasive form of breast cancer (BC) requires a sentinel lymph node biopsy (SLNB). In some patients, a pathological examination of dissected lymph nodes during SLNB reveals a presence of metastatic lesions (SLN+). The current standard for treatment of SLN(+) BC patients allows to spare lymph nodes without a need to perform an additional axillary lymph node dissection (ALND) in selected patients7.The Z0011 study initiated an era of conservative treatment in selected SLN(+) patients. The study also included cases with limited, however not specified ECE of metastatic SLN11 and in 21.7–57.5% of nSLNs13. ECE is an important prognostic factor with negative effect on the overall survival (OS), and recurrence-free survival (RFS)13.An extracapsular extension is present in 22–55% of metastatic SLN14. ECE exceeding 2 mm increases risk for a local and a regional recurrence of the disease by 27% and 33% respectively. Consequently, ECE not exceeding 2 mm presents with significantly lower risk of local and regional recurrence—18% and 22% respectively.The extracapsular extension classification proposed by Katz et al. is based on two levels of lesion severityThe aim of this study was to evaluate the clinical relevance of ECE in SLN in BC patients who underwent SLNB.Between January 2007 and December 2017 a total of 5223 BC patients underwent SLNB at The Department of Clinical Breast Cancer and Reconstructive Surgery, Oncology Centre—prof. Franciszek Łukaszczyk Memorial Hospital in Bydgoszcz. The retrospective analysis covers a group of 1145 SLN(+) patients. The need for informed consent from patients was waived by the Ethics Committee at the Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz . 908 of them underwent completion ALND), 891 cases presented ECE in SLN. The analysis did not include 237 SLN(+) patients receiving conservative treatment without ALND. The remaining exclusion criteria included: pre-invasive BC, bilateral BC, neoadjuvant treatment or previously diagnosed different type of cancer. The design of the study is shown in Fig. 4.Surgery and adjuvant treatment: radiation or systemic  were performed according to current recommendations for BC treatmentStatistical analysis covered selected clinical and pathological data: primary tumour size, grading, histological and biological type of cancer, type and number of metastases to SLN and non-sentinel lymph nodes, the presence and size of ECE.A three-level scale was used to describe the ECE presence and size. Three groups of patients were established: group I: no extracapsular extension in SLN; group II: focal extracapsular extension in SLN, up to 1 mm; group III: extensive extracapsular extension in SLN, above 1 mm , and patients with ECE—ECE(+). The ECE size (group II vs group III) were additionally evaluated.All patients underwent a complete surgical treatment at single institution. SLNBs, ALNDs were performed by experienced BC surgeons in the same way during the study.99mTc with 75–100 MBq, administered on an albumin carrier (Nanocol) was used to identify the SLN. At the beginning of the study, a combined method  was used to localize SLN. 34 of analysed patients underwent SLNB with dual technique.The isotope method, using 15, along with every intraoperatively palpated and clinically suspicious LN.The SLN was identified as a lymph node with the highest level of radiation (and/or containing the dye). In accordance with so called “Rule of 10%”, the LNs emitting radiation at a level exceeding 10% of the radio tracer emission value obtained for the SLN, were additionally resectedThe complete ALND (levels I–III) was performed in 891 patients.www.statistica.io, version 13.3). Relationships between categorical variables were assessed using the Chi-square test. The Student’s t-test or the Mann–Whitney’s U-test were used to compare continuous variables, depending on parameters of a distribution of a variable between groups.Statistical analyses were conducted using Statistica  was defined as a time between the surgery (SLNB) date and patient’s death (regardless of its cause). The recurrence-free survival (RFS) was defined as a time between SLNB date and diagnosis of the first recurrence . The overall survival time and the recurrence-free survival time were assessed using the Kaplan–Meier model, and compared using the log-rank test. In all statistical analyses, the cut-off value for the probability coefficient was set at p value of ≤ 0.05.Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) in predicting ECE in nSLNs(+) was determined.The last follow-up visit was conducted in June 2019.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performer by any of the authors. The study was approved by the Ethics Committee at the Nicholas Copernicus University in Toruń, Collegium Medicum in Bydgoszcz .In 433 (48.6%) patients, cancer metastases were limited to the SLN (group I), while in 61 (6.8%) patients the SLN capsule exceeded focally (group II). In the remaining 397 (44.6%) patients, a more extensive infiltration to the perinodal area was confirmed (group III). The average patient age was 57.0 years (ranging from 23 to 89 years).The most common type of metastatic lesions in SLN in the ECE(−) group were macrometastases (86.6%), followed by micrometastases 12.7%). In the ECE(+) group, macrometastases and micrometastases were found in 97.6% and 2.4% of patients, respectively (p < 0.0001). Metastatic lesions to nSLNs were diagnosed in 27.0% of patients from group I and in 48.9% of the patients with ECE (p < 0.0001). Similar differences also concerned the pathological evaluation of LNs , as well as a number of metastatically changed LNs  patients and in 14.6% of ECE(+) patients (p = 0.0251). Luminal A BC was diagnosed in 37.9% of ECE(−) patients and in 45.9% of ECE(+) patients (p = 0.016). Detailed data on clinical and pathological outcomes of patients included in the study is shown in Table ECE was found in 42.6% (234/550) of the nSLNs(−) patients and in 63.7% (227/341) of the nSLNs(+) patients (p < 0.0001).The focal extracapsular extension in SLN was reported in 6.2% (34/550) of the nSLNs(−) and in 7.9% (27/341) of the nSLNs(+) patients (p = 0.3175), while extensive ECE was observed in 36.4% (200/550) and 57.8% (197/341) of nSLNs(−) and nSLNs(+) patients, respectively (p < 0.0001).Sensitivity, Specificity, positive predictive PPV and NPV in predicting ECE in nSLNs(+) patients were 65.7%, 57.5%, 48.9%, 73%, respectively.The median time for patient follow-up was 67 months (18–152 months). A recurrence of underlying disease was diagnosed in 96 (10.8%) patients. Among them, the local, regional, and distant cancer recurrence was found in 21.9%, 4.2% and 79.2% of patients, respectively.In total, 74 deaths were reported. Detailed data on long-treatment outcome is presented in Table This study evaluated clinical relevance of ECE in BC metastases to SLN. Among available reports, our study analysed the largest number of cases from a single institution. The adopted method for data collection ensured an optimum consistency of the analysed clinical material.9. When compared to nSLNs(−) patients, infiltration to SLN perinodal tissues in the nSLNs(+) group was found in 71.4% patients when compared to the entire cohort. ECE proved to be even more clinically significant than the character (micro- or macrometastases) of metastatic lesions in SLN. Consequently, ECE allowed development of statistical tools for prediction of metastatic lesions in nSLNs(+) patients17. Our study indicates that ECE statistically increase percentage of nSLNs(+) cases when compared to ECE(−) patients, which is in accordance with previous reports18. Nonetheless, we did not find any significant influence of the ECE size on other variables related to the stage of metastatic lesions in the lymphatic system. It should be emphasised that the pN stage and number of metastatic LNs differed significantly between ECE(−) and ECE(+) groups.As demonstrated previously, the ECE was the most important variable increasing risk of the metastatic lesions in nSLNs8. Of the variables compared by the authors, significant differences concerned the pN stage, type of metastatic lesions in SLN, and the size of primary tumour.Similar results were presented by Schwentner et al., where 343 SLN(+) patients, including 104 cases of ECE(+) were analysed12, ECE is more frequent in patients with primary multifocal lesions, invasion of lymph vessels, presence of steroid hormone receptors and large tumours.According to the results of the studies conducted by Gooch et al.Our study did not demonstrate the influence of the ECE on long-term treatment results. Nevertheless, patients with ECE were more frequently qualified for adjuvant CHTH and adjuvant RTH when compared with ECE(−) patients . Noteworthy, the differences in RTH administration were not influenced by the surgical treatment, since BCT rate was comparable in both analysed groups. This could therefore influence the achieved treatment results.8. Similar results were also presented by Drinka et al.5 and Shigematsu et al.7. However, in SLN(+) patients, ECE is considered as an independent DFS prognostic factor19.In a study conducted by Schwentner et al., different treatment results were achieved. The ECE presence was associated with worse 5-year OS, but it had no influence on 5-year DFS20. However, due to retrospective nature of the studies, lack of the complete evaluation of the axillary treatment on long-term outcomes and relatively small patient groups, these results should be interpreted with caution.Similar observations concern the extent of ECE. ECE below 2 mm has no influence on the recurrence rate, and is similar to ECE(−) patients23, unambiguous evidence for management of patients with ECE is not available. However, since significant differences in long-term treatment outcomes in ECE(+) patients were shown, further prospective studies are warranted.Despite gradual limiting of indications for surgical treatment in SLN(+) patientsThis study contains certain limitations. Due to the three-level method for ECE size evaluation , as well as a retrospective character of the study, only three groups of patients could be distinguished. This significantly limited the scope of statistical calculations, including determination of other groups of patients.Although with no effect on long term treatment outcomes, ECE in SLN + BC patients is accompanied by higher pN stage and therefore seems to be an justified indication for ALND and complementary adjuvant treatment.


TrFM/ASM was not associated with TL. Preliminary findings suggest that sarcopenia and obesity may act synergistically to shorten telomeres.Sarcopenic obesity (SO) is characterised by the concurrent presence of sarcopenia and excess adiposity. Telomere shortening has been associated with sarcopenia and obesity alone but the association between SO and telomere length (TL) has not been investigated. This study aimed to investigate SO and TL in an adult population. Data were from 5397 individuals  enrolled in the National Health and Nutrition Examination Survey. Body composition (BC) was assessed by Dual Energy X-Ray Absorptiometry. Two models were used to assess SO: a BC model including four phenotypes derived from the combination of high or low adiposity and muscle mass; and, a truncal fat mass to appendicular skeletal mass ratio (TrFM/ASM). TL was assessed using quantitative polymerase chain reaction and expressed as base pairs. The mean TL, relative to the reference DNA, was calculated and expressed as the mean T/S ratio. A General Linear Model was applied to determine associations between TL for SO. In adjusted analysis, only individuals with SO, defined as the presence of high adiposity-low muscle mass (four-phenotype model), had significantly shorter telomeres ( Sarcopenic obesity (SO) is defined by the concurrent presence of low muscle mass (sarcopenia) and high adiposity (obesity) . SO appeTelomeres are DNA-protein structures located at the ends of chromosomes, which protect the genome from inter-chromosomal fusion, nucleolytic degradation, and genome instability . In the Therefore, the aim of this study was to explore the relationship between SO and TL in an adult population-representative cohort recruited as part of National Health and Nutrition Examination Survey (NHANES).http://www.cdc.gov/nchs/nhanes.htm (accessed March 2021). The survey used a complex, multistage probability sampling design to ensure that the sample was representative of non-institutionalised adult (≥18 years) civilians in the United States (US) [n = 1712) or had a coefficient of variation for mean T/S ratio (mean T/S ratio/standard deviation of mean T/S ratio) that was >1 (n = 1), were excluded. The final sample included 5397 individuals.The data for this study were obtained from the NHANES surveys conducted between 1999 and 2002. Survey procedures are available online at tes (US) . The samBody composition (BC) was assessed by DXA using a Hologic QDR-4500A. Participants were excluded if they were pregnant or they did not fit on the DXA scanner (weight > 136.1 kg and height > 195.6 cm). Participants were also excluded if they had been exposed to nuclear medicine in the previous three days or radiographic contrast material in the previous seven days.2 (m). Participants were classified as normal weight (BMI = 18.5–24.9 kg/m2), overweight (BMI = 25.0–29.9 kg/m2) or obesity (BMI ≥ 30.0 kg/m2).Body composition phenotypes were defined using three separate models: (1) a four-phenotype model; (2) the ratio between truncal fat mass (TrFM) and appendicular skeletal muscle mass (ASM); and (3) body mass index (BMI). The four-phenotype model divides individuals into four categories based on levels of adiposity and muscle mass, and previously established reference curves . The fouTelomere length was analysed using whole blood and qPCR and reported as base pairs. The mean TL, relative to the reference DNA, was calculated and expressed as the mean T/S ratio. Assay runs with control values beyond two standard deviations of the mean of all runs, was excluded (<6% of runs). Outliers within samples were identified and excluded (<2% of samples). The interassay coefficient of variation was 6.5%.https://wwwn.cdc.gov/nchs/nhanes/tutorials/Module4.aspx (accessed March 2021). Analysis was prepared by adjusting for strata, clusters, and a 4-year sample weight. Descriptive statistics were calculated through complex analysis unless otherwise stated, and presented as mean with standard error (continuous variables) or as percentages of total sample weight . A General Linear Model (GLM) was applied to the three models of BC, with the mean T/S ratio selected as the outcome variable on each occasion. Analyses were adjusted for age, sex, ethnicity, education, physical activity, smoking, alcohol intake and general health condition, which are covariates that have been described previously [p value ≤ 0.05 was considered as statistically significant.Statistical analysis was conducted using the SPSS Complex Samples module according to NHANES protocols found online at eviously . The GLMth centile (SO). The BMI model classified 33.8% of individuals as normal weight, 36.0% as overweight, and 29.6% as individuals with obesity . The four-component BC phenotype model classified 25.0% of individuals as LA-HM, 24.7% as LA-LM, 27.9% as HA-HM and 22.4% as HA-LM (SO). The TrFM/ASM model classified 14.7% of individuals as <15th centile, 70.5% as 15–84th centile and 14.7% as ≥ 85.3% male.p = 0.05) compared to 1.05 (95%CI: 1.01–1.09), respectively. The mean T/S ratio for the HA-LM group corresponded to a TL of 5735 bp (95%CI: 5650–5820), which was significantly shorter  compared the reference group LA-HM. No significant association was found between TrFM/ASM phenotypes and TL, or between BMI groups and TL  phenotype and TL whereas no significant association was found between BMI groups and telomere shortening. These findings are contrasting with previous literature indicating a significant association between BMI and telomere shortening; a potential reason for the difference could be attributed to the difference in sample size as the previous analysis was based on eighty-seven distinct study samples were including 146,114 individuals , 13.This study has numerous strengths. This analysis included a large number of individuals that were multi-ethnic and representative of the adult civilian population in the US. BC was assessed using DXA, which is currently the preferred field method of assessment based upon accuracy and reliability. The analyses were also adjusted for demographic, health and lifestyle covariates. TL was measured by a well-established protocol, which included stringent quality control measures such as the identification and exclusion of outlying values. However, this study also has limitations. Subjects were excluded from DXA-assessment if they weighed over 136.1 kg or were taller than 195.6 cm, which may render these findings unrepresentative of those with extreme body types. The classification of sarcopenia did not include the assessment of muscle function, which is a distinct component of sarcopenia assessment. It was not possible to investigate a causal association between BC phenotypes and telomere shortening due to the cross-sectional design of the study.In conclusion, these preliminary results indicate a significant association between SO and telomere shortening and may suggest that sarcopenia and obesity may act synergistically on the ageing process. Future work should be conducted to further explore these associations in longitudinal cohorts to understand whether SO may be associated with a greater rate of telomere shortening.