Population aging is a looming global health challenge. New biological aging metrics based on DNA methylation levels have been developed in addition to traditional aging biomarkers. The prospective relationships of aging biomarkers with mitochondrial changes are still not well understood. Here, we examined the prospective associations of mitochondrial copy number (mtDNAcn) with several aging biomarkers – DNAm-Age, DNAm-PhenoAge, DNAm-GrimAge, and leukocyte telomere length. We analyzed 812 individuals from Veteran Affairs Normative Aging Study (NAS) with available blood samples from 1999-2013. Whole blood mtDNAcn and relative leukocyte telomere length were measured via qPCR. DNA methylation was assessed and used to calculate DNAm-Age, DNAm-GrimAge, and DNAm-PhenoAge. Linear mixed models were used to quantify the associations of mtDNAcn with DNAm-Age, DNAm-GrimAge, DNAm-PhenoAge, and leukocyte telomere length. In multivariable cross-sectional analyses, mtDNAcn is negatively associated with DNAm-Age PhenoAge and DNAm-PhenoAge. In contrast, mtDNAcn is associated with prospective measures of higher DNAm-PhenoAge and shorter leukocyte telomere length. Our study shows that higher mtDNAcn is associated with prospective measures of greater DNAm-PhenoAge and shorter leukocyte telomere length independent of chronological age. This indicates a role for mitochondrial in aging-related disease and mortality, but not the departure of biological age from chronological age. Life expectancy is projected to continue to increase across the globe [In 2015, 8.3% of the global population was older than 65 years of age, but as the world population ages, this number is estimated to grow to 15.8% by 2050 [Telomere length is a well-known aging biomarker. Telomeres shorten with age in a broad range of organisms  and shorUnfortunately, the underlying biological and molecular processes that drive these epigenetic age biomarkers are still unknown. Indeed, there is still a lack of understanding about what they represent on a molecular level. A recent review  pointed Mitochondrial DNA copy number (mtDNAcn), a measure of mitochondrial genome abundance, is commonly used as a reflection of the mitochondria’s response to oxidative stress as well as general dysfunction . MitochoRecently, our group has shown that cross-sectionally, mtDNAcn is negatively correlated with DNAm-Age and hypothesized mtDNAcn may be a proxy of mitochondrial buffer capacity . Reduced. The mean leukocyte telomere length at baseline was 1.3 (±0.5). DNAm-Age was weakly correlated with DNAm-PhenoAge (r=0.33) and DNAm-GrimAge (r=0.29), but DNAm-PhenoAge and DNAm-GrimAge were strongly correlated (r=0.77) ((r=0.77) . TelomerAdvancing chronological age was associated with lower mtDNAcn  and shorter leukocyte telomere length  and increased DNAm-Age , DNAm-PhenoAge , and DNAm-GrimAge  .First, we sought to extend our previous analyses  using adNext, we examined whether baseline mtDNAcn is associated with the aging biomarkers at all future visits by modeling the baseline measure of mtDNAcn with the aging biomarkers measured during subsequent follow-up visits . In multTo investigate possible presence of reversal causality, we examined whether baseline DNAm-Age and baseline DNAm-PhenoAge were associated with prospective measures of mtDNAcn. There were no associations between baseline DNAm-Age and DNAm-PhenoAge with prospective measures of mtDNAcn (data not shown). Additionally, we tested the robustness of our models by excluding those who were diagnosed with CHD or diabetes, those who reported active smoking, or those who were not self-reported as white. In all cases, the resulting effect estimates were not meaningfully different compared to the results in In our analysis of 812 aging male veterans from the greater Boston area, we found contrasting results between cross-sectional and prospective analyses of mtDNAcn with aging biomarkers DNAm-Age, DNAm-PhenoAge, DNAm-GrimAge and leukocyte telomere length. Overall, we found suggestive evidence that mtDNAcn is positively associated with prospective measures of DNAm-PhenoAge and negatively associated with prospective measures of leukocyte telomere length. These observed associations were independent of chronological age, suggesting that higher mtDNAcn is associated to greater biological aging.The results of our cross-sectional analyses presented here and in our previous report  are geneOur prospective analyses stood in contrast to the cross-sectional results. Adjusting for measures of aging at baseline, mtDNAcn was positively associated with prospective measures of DNAm-PhenoAge. Similarly, although mtDNAcn was not associated with leukocyte telomere length cross-sectionally, there was an negative association between mtDNAcn and leukocyte telomere length at follow up visits, which is in accordance with studies that have previously reported that mtDNAcn is associated with shorter telomere length , 35. TheWhile we observed associations of mtDNAcn with DNAm-PhenoAge and leukocyte telomere length, no associations were observed with DNAm-Age and DNAm-GrimAge. This disparity may be due to the differences in the underlying biological drivers behind each of the aging metrics. DNAm-Age is based on chronological age and data obtained from multi-tissues samples and aims to reflect the general aging process. Specifically, it is thought that while DNAm-Age is associated with age-related diseases, it does not fully capture the risk differences for death and disease . In contCurrently, the underlying biological relationships between mitochondrial health, as reflected by mtDNAcn, and biological aging are unclear. It is possible that mitochondrial plays a role in aging-related biology and aging-related diseases either independently of mechanisms that affects both mitochondrial health and aging or as a mediator. Biologically, the mitochondrial genomic content (i.e. mtDNAcn) can increase in response to stress via mitochondrial biogenesis to enhance energy supply and repair damage to cellular components . It is pa priori knowledge of biological/clinical relevance to select and control for potential confounders, but we cannot rule out the possibility of unknown or residual confounding from underlying biological processes or external factors such as physical activity and psychosocial stress. In this scenario, mtDNAcn is a reflection or product of physical activity, psychosocial stress, or some underlying biological process that also causes biological aging or aging-related diseases and mtDNAcn may not be directly associated with higher DNAm-PhenoAge or lower leukocyte telomere length.To our knowledge, our study is the first to report on the prospective associations of mtDNAcn with aging biomarkers DNAm-Age, DNAm-PhenoAge, DNAm-GrimAge and leukocyte telomere length. The current study possesses a number of strengths including the use of a large longitudinal cohort with repeated measures of aging and mitochondrial biomarkers. However, our study also has limitations. Our cohort is comprised primarily of white older men living in New England. There are other differences in the measures of biological aging by demographic factors (e.g. sex) that we were not able to examine. Thus, additional studies involving other demographic groups in different environments will be needed to confirm our findings more broadly. Finally, we used the existing literature and Our study of 812 men from an aging cohort indicate the involvement of mitochondria in modulating biological aging, as reflected by DNAm-PhenoAge and leukocyte telomere length. Overall, we found evidence that higher mtDNAcn may be associated with higher DNAm-PhenoAge, indicating increased risk for aging-related disease and mortality, but not necessarily with departure of biological age from chronological age. Currently, the biological relationships between mitochondrial health and biological aging are not fully understood and future studies are necessary to further clarify the breadth of interactions between the mitochondria and aging biomarkers in human aging and to confirm our findings in other populations.The Veteran Affairs Normative Aging Study (NAS), a closed longitudinal cohort study of men from the Greater Boston area, was established in 1963 with 2280 individuals and followed up every 3-5 years. At enrollment, all participants were veterans 21-80 years of age, lived in the greater Boston area, and were free of chronic diseases. During follow-up study visits they undergo comprehensive outpatient medical evaluations and provide detailed data regarding diet and other lifestyle factors. Whole blood was collected after overnight fasting from each participant during NAS follow-up visits. The present study comprises 2186 visits from 812 subjects with available blood samples from 1999 to 2013 where 596 subjects had at least 1 follow-up visit and 438 had >2 follow-up visits.Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for mtDNAcn using total whole blood DNA as previously described , 41. DNAhttps://dnamage.genetics.ucla.edu/home). The DNAm-PhenoAge has been calculated based on methods described by Levine et al. [DNA methylation from whole blood DNA extracted from the buffy coat layer was interrogated using the Infinium HumanMethylation450 BeadChip (Illumina). To minimize batch effects and ensure a similar age distribution across chips and plates, we randomized sample across plates and used a two-stage age-stratified algorithm to randomize samples. We pre-processed the samples with Illumina-type background correction without normalization, corrected for probe types using the BMIQ method , and reme et al. . Lastly,e et al. Leukocyte telomere length assay was performed using qRT-PCR as described –47. In bA total of 812 participants had available blood samples from visits between 1999 and 2013. There were no differences in baseline characteristics between those included in our analyses compared to those excluded due to lack of biological samples. Outliers for mtDNA, DNAm-Age, DNAm-PhenoAge, and leukocyte telomere length were removed if the values were >3 SD from the mean.We used random intercepts linear mixed models (LMMs) to study relationships mtDNAcn and the aging biomarkers. We first conducted cross-sectional analyses of mtDNAcn with aging biomarker outcomes where mtDNAcn from all visits were modeled with aging biomarkers from the same visits. Then, as prospective analyses, mtDNAcn from the first visit (i.e. earliest visit since 1999) was modeled as the exposure while the prospective measures of aging biomarkers from following visits were modeled as outcomes. For all models, mtDNAcn was modeled as both quartile and continuous variables. In the multivariable models, we controlled for the following covariates based on biological plausibility and previous works : chronolTo examine the potential for reverse causation whereby greater biological aging may be driving mitochondrial changes, we examined the associations of baseline DNAm-Age, DNAm-PhenoAge, and leukocyte telomere length with prospective measures of mtDNAcn. For sensitivity analyses, we re-ran all analyses restricting to those without diabetes or CHD, non-active smokers, and only those who self-reported as white.Analysis was performed with R (v3.5.2) (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.) using the ‘lme4’ package. Statistical significance was defined as p-values < 0.05.Supplementary Figure 1Supplementary Tables


Few studies examining the impact for women of employment status on health have considered domestic duties and responsibilities as well as household socioeconomic conditions. Moreover, to our knowledge, no studies have explored the influence of work-family conflict on the association between employment status and health. This research aimed to investigate the cross-sectional associations between employment status  with self-rated health among Japanese middle-aged working women.Self-report data were obtained from 21,450 working women aged 40–59 years enrolled in the Japan Public Health Center-based Prospective Study for the Next Generation (JPHC-NEXT Study) in 2011–2016. Multivariate odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for poor self-rated health (‘poor’ or ‘not very good’) by employment status. Sub-group analyses by household income and marital status, as well as mediation analysis for work-family conflict, were also conducted.Adjusted ORs for the poor self-rated health of non-regular employees and self-employed workers were 0.90  and 0.84 , respectively, compared with regular employees. The identified association of non-regular employment was explained by work-family conflict. Subgroup analysis indicated no statistically significant modifying effects by household income and marital status.Among middle-aged working Japanese women, employment status was associated with self-rated health; non-regular employees and self-employed workers were less likely to report poor self-rated health, compared with regular employees. Lowered OR of poor self-rated health among non-regular employees may be explained by their reduced work-family conflict. Further, the voluntariness of a job choice for non-regular employees may differ based on household socioeconomic conditions and may be influenced by varying societal norms regarding gender roles.18One of the possible reasons for the inconsistent results could be underlying differences in participants’ reasons for being temporary workers.19 The gender role norms  that exist in Japanese society could be one of the reasons for this significant gender difference. Women’s participation in Japan’s workforce by age group shows a bimodal pattern. By contrast, similar to Japan’s male workforce, many Western societies display a convex shape.20 This bimodal pattern suggests that women tend to take a career break during their 20s and 30s, for such reasons as child rearing, and return to the workforce in their 40s.21,22 Part-time work is often the only choice for women who reenter the workforce23; it is an important path for women reentering the labor market.24 Consequently, a much higher proportion of women than men in Japan are non-regularly employed (mainly part-time).19In Japan, the number and proportion of female non-regular workers  is much larger than that of non-regular male workers. In 2018, 54% of female employees and 14% of male employees aged 24–65 years old were non-regular workers.25 However, these promotional efforts have not improved the imbalanced participation in household duties. That is, in most societies including Japan, women’s household responsibilities have persisted despite more women entering and playing a greater role in the workforce.26–28 Work-family conflict, generally defined as “a form of inter-role conflict in which the role pressures from the work and family domains are mutually incompatible in some respect,”29 has been associated with women’s self-rated health.30–33 An international comparison study showed that Japanese female workers had the highest work-family conflict and poorest self-rated mental and physical health among Japanese, Finnish, and English government workers.34 Other research, moreover, has shown that the working arrangement is associated with the prevalence of work-family conflict, with higher work-family conflict existing among full-time versus part-time workers.33,35 Women in Japan are often obligated to work part-time to mitigate their burden of managing both work and household demands and responsibilities because non-regular employment often offers more schedule flexibility compared with regular employment.36,37 Thus, we hypothesized that the association between employment status and self-rated health may be explained by the level of work-family conflict. Indeed, one of the main reasons middle-aged women in Japan choose non-regular employment and self-employment is time flexibility,19 which can reduce work-family conflict raised by multiple social roles for working women.38 To our knowledge, no studies have explored the effect of work-family conflicts on the association between employment status and health.The proportion of female non-regular workers has increased along with the promotion of women’s participation in the workforce in the last few decades in Japan.18 Thus, household socioeconomic conditions may impact the association between employment status and health.26–28 However, few studies have included domestic duties and responsibilities and household socioeconomic conditions in examinations of the impact of employment status on health among women.Available financial and material resources can affect the conditions relating to employment status. For example, some women work part-time to make a living, while others only need to boost the household budget; such differences produce heterogeneity amongst non-regular employees.The current study thus aimed to investigate the associations of employment status  with self-rated health among Japanese middle-aged working women. Our specific research questions were:1) Is employment status associated with the probability of having poor self-rated health among Japanese working women?2) Is the association between employment status and self-rated health explained by level of work-family conflict?3) Are the associations between employment status and self-rated health noted above modified by socioeconomic conditions?39Data in this study were derived from the Japan Public Health Center-based Prospective Study for the Next Generation (JPHC-NEXT Study). The JPHC-NEXT study was initiated in 2011 and the baseline survey was completed by December 31, 2016. In 2011–2016, we established a population-based cohort of 261,939 residents aged 40–74 years who registered an address in cities, towns, and villages  in seven prefectures throughout Japan. A self-administered questionnaire was distributed mostly by hand and partly by post to all participants; questions were asked about their lifestyles, personal medical histories, and socio-demographics. Incomplete answers were followed up by telephone interview. Among 261,939 residents, 115,385 agreed to participate in the JPHC-NEXT Study . All participants provided written informed consent. We excluded those with did not respond to the questionnaire. Our final cohort population was 114,157 .The JPHC-NEXT Study was approved by the institutional review boards of the National Cancer Center and other participating institutions.n = 24,375). From these, we excluded 1,778 women with a history of cancer or cardiovascular disease and/or physical limitations; we excluded a further 1,147 women with undisclosed employment status, self-rated health, and/or work-family conflict scores. The final study population comprised 21,450 women.We excluded women aged 60 years and older, women who were unemployed, and women for whom no information on occupation was available, to restrict our study population to working women 40–59 years old (Our main predictor variable was employment status. Employment status was identified using self-report and categorized into three groups: 1) regular employee, 2) non-regular employee , and 3) self-employee.40,41 we categorized the responses into two groups: 1) poor self-rated health (‘poor’ or ‘not very good’), and 2) not poor self-rated health .We measured self-rated health as our outcome variable, using the single questionnaire item: “How would you describe your overall state of health?”. Participants chose one of five responses: poor, not very good, good, very good, and excellent. Following previous studies,42 Equivalent household income was classified into quintiles; we also categorized equivalent household income into two groups by its median (High and Low) for sub-group analysis.Age; highest education attainment level ; occupation category ; equivalent household income (quintile); marital status (married and non-married); history of hypertension, diabetes mellitus, hypercholesterolemia, and any other diseases ; and residential area were hypothesized as confounding factors. With regard to income, we obtained the data for annual household income using six categories. We calculated household equivalent income by dividing household income  by the square root of total household members.43,44 The questionnaire for work-family conflict consisted of eight items . The responses to the eight items were summed up to yield a measure for work-family conflict scores ranging from 0 to 16. The internal consistency of work-family conflict in this study population was deemed acceptable . Further evidence on the reliability and validity of these scales have been provided by several other studies.33,34 The scores for work-family conflict were grouped into tertiles for the analysis in accordance with previous studies.34The data for work-family conflict was obtained through a self-reported questionnaire. Previous studies suggest that work-family conflict consists of two factors (work-to-family and family-to-work conflicts), which can be combined as a joint scale.We calculated the number (%) or mean (standard deviation [SD]) of the study population’s characteristics. Using chi-square tests and analysis of variance, differences among employment statuses were calculated in terms of proportion and mean values for poor self-rated health, socioeconomic variables, marital status, medical history of hypertension, diabetes mellitus, or hypercholesterolemia, history of diseases, and work-family conflict scores.45 We also estimated the percentage of the total associations between employment status and self-rated health that were mediated through work-family conflict using log ORs.46Odds ratios (ORs) with 95% confidence intervals (CIs) of employment status for poor self-rated health were calculated using logistic regression analysis after adjusting for hypothesized confounding variables, such as age, education level, household equivalent income, occupation, marital status, medical history of hypertension, diabetes mellitus, hypercholesterolemia, or any other diseases, and residential area (model 1). To explore the mediating effect of work-family conflict on the association between employment status and self-rated health, we included work-family conflict scores in the model (model 2). Further, we conducted mediation analysis and estimated direct and indirect effects of employment status on self-rated health through work-family conflict. The natural direct effect (NDE), natural indirect effects (NIE), and total effects (TE) were estimated as ORs for poor self-rated health with respect to the mediator, conditioned on the measured covariates.Sub-group analyses by household equivalent income and marital status were also performed for self-rated health. We tested statistical interactions using cross-product terms for employment status and household equivalent income or marital status. All analyses were performed using SAS version 9.4 .Table Table The results of mediation analysis by work-family conflict scores showed that the association between non-regular employment and self-rated health was largely mediated through work-family conflict (the proportion mediated through work-family conflict was 100%), and no direct effect of non-regular employment on self-rated health was identified Table . Part .17 Although only a limited number of similar studies have been conducted in Japan, our results were consistent with those of the Comprehensive National Survey among 18–64-year-old Japanese female employees. They reported that, compared with permanent employment, unstable employment with short working hours was associated with a smaller proportion of women who rated their health as poor.47Our results are in line with other research findings24 Thus, working as regular employees often leads to severe physical and psychosocial hardship for women.38 Women in Japan who were regular employees were more likely to report job pressures and inflexible work schedules and to experience more strain related to work and family than their non-regularly employed counterparts.48 Moreover, Japanese female workers were reported to have the highest work-life conflict and poorest self-rated mental health among Japanese, Finnish, and English government workers.34 Thus, female workers in Japan may voluntarily choose to work part-time to achieve a balance between their work and family lives.One of the reasons for poorer self-rated health among regular employees compared with non-regular employees could be the difficulties that women in Japan face in reconciling family life and career. Female workers in Japan who play a significant part of the total workforce are also more likely to be influenced by strong societal gender norms to fulfill household duties.Our results  suggest that non-regular employees may have achieved better self-rated health than regular employees by buffering their work-family conflict. The possible benefits of non-regular employment, such as schedule flexibility and/or fewer duties and responsibilities at work, can reduce their work-family conflict, which may contribute to their reduced probability of having poor self-rated health. Our findings also suggest that Japanese female workers may find relief from difficult life situations caused by juggling work and family lives by taking non-regular jobs or being self-employed, which may also be beneficial for their self-rated health.5 While we do not have a clear explanation for this inconsistency, we speculate that one possible reason could be differences between two outcomes in the mechanisms of the health impact of employment status. That is, self-rated health reflects current life conditions on a short-term basis, while mortality may reflect chronic and continued effects relating to employment such as job insecurity, financial insecurity, and social welfare. These chronic health effects may be particularly strong in Japan, where there is little employment flexibility and the gap between regular and non-regular employees is relatively large.24 In other words, being a non-regular employee may alleviate the psychosocial and physical burdens temporarily; however, it may lead to long-term health deterioration through chronic psychosocial and socioeconomic impacts. In addition, we identified that work-family conflict explained most of the association between employment status and the risk of having poor self-rated health in this study. Controlling for the influence of work-family conflict, we identified no significant differences in probabilities of having poor self-rated health by employment status. In other words, most of the differences observed in self-rated health by employment status may be attributed to differences in psychological well-being. We do not deny the effect of psychological health on mortality; however, we speculate that the lower risk of self-rated health among non-regular employees, mostly explained by psychological health, may not directly reflect the results for mortality.Our results were, however, inconsistent with previous work that showed a higher mortality risk for non-regular female employees than for regular employees in Japan.49 In contrast to non-regular employees, work-family conflict did not appear to explain the identified association between self-employment and poor self-rated health. Work-family conflict is constructed to show the tension between two mutually incompatible domains, work and family.29 The work and family lives of self-employed workers may not present as much conflict as those of the other types of employees because the two domains (work and family) are physically closer and there is greater job autonomy. Further research is needed to understand the mechanisms of the association between being self-employment and self-rated health.Self-employed workers also showed better self-rated health compared with regular employees. Job autonomy, job control, and/or control of working hours are hypothesized as possible benefits of being self-employed.24Although we did not identify statistically significant interaction of socioeconomic conditions and employment status on self-rated health, the benefits of being a non-regular employee or being self-employed was greater among women with higher socioeconomic status . Married women and women with a higher socioeconomic background are, arguably, more likely to be working to add extra income to that of the other breadwinner in the household. Thus, they can enjoy the flexibility of non-regular work, and, importantly, take advantage of it by reducing work-family conflict. By contrast, unmarried women and women with in lower socioeconomic conditions are more likely to be making their own living, which could make them vulnerable to the disadvantages of non-regular employment, such as low payment and poor social security.This study is one of the few to observe the association between employment status and self-rated health in Japan and to explore the associations that are related to work-life conflict. However, there are several limitations. First, given the cross-sectional nature of the study, we cannot claim causative links; in particular, we cannot exclude the possibility of reverse causation. To reduce this possibility, we excluded women with medical histories of major diseases and statistically controlled the medical history of diseases. We also conducted sensitivity analysis by further excluding women with medical histories of diseases, which did not change our conclusions . Further research is required to establish causative links. Second, our results may have been affected by selection bias caused by non-participation or exclusion because of missing values in our main variables. For example, if non-participation occurred disproportionally in self-rated health conditions or in employment status, our conclusions could have been distorted. We do not have specific information about the direction of bias. However, excluded women with missing values on self-rated health, employment status, and work-family conflict were likely to have lower socioeconomic status, which implies that our results may be underestimated. Third, our study population may not be nationally representative; in particular, we did not include metropolitan areas. In addition, our study population was limited to women aged in their 40s and 50s. They may be widely different from younger generation in terms of their life situations, life-styles, domestic duties and responsibilities, and work-life balance. Thus, the generalizability of our results to other populations requires caution. Finally, measurement errors in our variables, including our outcome and predictor and unmeasured (confounding) variables, may have resulted in residual confounding.Employment status is associated with probabilities of poor self-rated health among middle-aged working Japanese women; non-regular employees and self-employed workers were less likely to report poor self-rated health compared with regular employees. Additionally, most of the associations with the poor self-rated health of non-regular employment could be explained by the level of work-family conflict. We did not identify significant influences of household income and marital status on the association between employment status and self-rated health.


Nature Communications 10.1038/s41467-020-19622-y, published online 13 November 2020.Correction to: https://adknowledgeportal.org). The AD Knowledge Portal is a platform for accessing data, analyses, and tools generated by the Accelerating Medicines Partnership (AMP-AD) Target Discovery Program and other National Institute on Aging (NIA)-supported programs to enable open-science practices and accelerate translational learning. The data, analyses, and tools are shared early in the research cycle without a publication embargo on secondary use. Data are available for general research use according to the following requirements for data access and data attribution . See: 10.7303/syn23262875.’In the original version of this Article, the Data Availability statement was incomplete. The following text has been added to the Data Availability statement: ‘The data described in this manuscript are available via the AD Knowledge Portal (


As advanced heart failure (HF) with elevated NT-proBNP is characterized by an activated coagulation system, coronary events clinically noticed as sudden or HF death may be more common after treatment with first- compared to newer-generation DES. Our study evaluates (1) if patients with left ventricular dysfunction (LVSD) who underwent percutaneous coronary intervention have a better survival with first- or newer-generation DES, and (2) if the survival benefit is predicted by NT-proBNP. Our observational study evaluated patients with LVSD who were registered in the coronary catheter laboratory database of the Medical University of Vienna. Multivariate Cox regression analyses tested an interaction in the risk of death between those with lower or elevated NT-proBNP levels and the stent-generation. The relative risk of newer- compared to first-generation DES as reference was calculated for patients with low and elevated NT-proBNP levels. In 340 patients (178 newer- and 162 first-generation DES) stent-generation and NT-proBNP were independent predictors of death. When the stent-generation*NTproBNP interaction was forced into a Cox regression model, this term independently predicted death. The relative risk of first- compared to newer-generation DES was similar in patients with lower NT-proBNP , but was higher in patients with elevated NT-proBNP . Death is associated to stent-generation. NT-proBNP is a predictor for the stent generation used: elevated levels demonstrated a higher mortality risk when using first- compared to newer-generation DES, while lower levels showed a similar risk when using either DES-generation. Nevertheless, remote follow-up data revealed higher rates of late and very late thrombotic events. Newer-generation DES were designed to overcome these long-term safety issues and, indeed, reduced stent thrombosis in the early and long term. However, these improvements in stent design like thinner stent-struts and improved tissue-compatibility of the polymer4. This prothrombotic shift seems to be more pronounced in more severe HF. Natriuretic peptides vary from normal levels to excessive elevation in patients with moderate to severe LVSD and, thereby, indicate different stages of disease5. Accordingly increasing levels of natriuretic peptides correlate with an increasing activation of the coagulation system as displayed by elevated D-dimer and von Willebrand Factor (vWF)6. In various clinical settings, higher levels of N-terminal proB-type Natriuretic Peptide (NT-proBNP) were related to thrombotic findings like thrombosis of transcatheter heart valves7 or spontaneous echo contrast as precursor for left atrial thrombosis8. In two pooled contemporary HF trials high NT-proBNP levels identified a subset of HF patients without atrial fibrillation at a high risk of stroke9.Heart failure (HF) based on left ventricular systolic dysfunction (LVSD) is associated with increased risk of stent thrombosis. This systemic disorder is accompanied by a prothrombotic environment due to an upregulation of prothrombotic and downregulation of antithrombotic factors10. However, autopsy studies reported coronary pathologies like plaque ruptures, intracoronary thrombi, and acute myocardial infarction to be frequent findings12. These coronary events are often clinically not recognized, but may trigger sudden or pump failure death. Due to the procoagulant state patients with ischemic cardiomyopathy and severe HF have an increased risk for stent-thrombosis and myocardial infarction13. Our study analyses if elevated NT-proBNP levels indicate worse outcome in patients with moderate to severe LVSD and coronary multivessel disease who were revascularized using first- compared to newer-generation DES.In most patients with ischemic heart failure the mode of death is clinically described as sudden cardiac or pump failure death. Especially when death occurs outside of the hospital, information is frequently lacking on prodromal symptoms; an electrocardiogram and cardiac biomarkers are rarely obtained14. In addition, NT-proBNP at time of hospital admission had to be available. At this time-point, all baseline characteristics were evaluated. The PCI was performed by experienced interventionists during the hospital stay of the diagnostic coronary angiography. Patients who underwent coronary artery bypass grafting (CABG) were excluded from analysis. Only adult patients with informed consent were included. CCLD-MUW provided the following data: baseline characteristics, co-morbidities, angiographic characteristics, blood results and echocardiographic parameter. The patients observational period started with initial coronary angiography and ended at the due day, which was the 31st of December 2014. All patients in < were managed according to the relevant heart failure guidelines of the European Society of Cardiology in one out of five heart failure clinics in Vienna. As there are no services such as telemedicine or homecare available for these patients in Vienna, additional multidisciplinary management was not possible for any of these patients. The survival status at due day was retrieved from the official Austrian death registry. The study was approved by the Ethics Committee of the Medical University of Vienna and is in line with the Declaration of Helsinki.We included adult patients from the coronary catheter laboratory database of the Medical University of Vienna (CCLD-MUW) between the years 2004 and 2012 in this observational cohort study. All patients suffered from significant CAD and LVSD. The definition of significant CAD was a stenosis of the left main stem ≥ 50% or major coronary artery ≥ 70% or 50–70% with objective evidence of myocardial ischemia and had to be present in at least one major coronary artery. For inclusion into the study left ventricular ejection fraction assessed by echocardiography (Simpson biplane) had to be below or equal to 40%15. Cox proportional hazards regression models were applied to identify independent predictors of death from any cause. These models included all variables that were univariate predictors of death except those which were available in less than 50% of patients . NT-proBNP levels were not normally distributed, therefore log transformed NT-proBNP levels were taken for continuous analysis within the Cox proportional hazards regression models. No methods came into operation to fill-up missing data or adjust the model for the presence of missing values16.Continuous variables were presented as median and interquartile range (IQR), categorical variables as counts or percentages. Continuous variables were presented by using an unpaired T-test, categorical data were presented by using crosstabs and chi-square test. Due to the relatively small study population and the exclusion of a significant number of unmatched patients with loss of according information when using propensity score matching we preferred covariate adjustmentModel A: To clarify if DES-generation (first or newer) is associated with death, death was adjusted by age, sex, rhythm, prior myocardial infarction, diabetes mellitus (DM), estimated glomerular filtration rate , Canadian Cardiovascular Society (CCS) class, New York Heart Association (NYHA) class, number of coronary vessels diseased, smoker, cerebrovascular and/or peripheral vascular disease, ejection fraction (EF) and DES-generation.Model B: To confirm an association between death and levels of NT-proBNP, death was adjusted by age, sex, rhythm, prior myocardial infarction, DM, eGFR, CCS class, NYHA class, number of coronary vessels diseased, smoker, cerebrovascular and/or peripheral vascular disease, EF and NT-proBNP.Model C: To identify an interaction between DES-generation and NT-proBNP, death was adjusted by age, sex, rhythm, prior myocardial infarction, DM, eGFR, CCS class, NYHA class, number of coronary vessels diseased, smoker, cerebrovascular and/or peripheral vascular disease, EF, NT-proBNP, DES-generation, and NT-proBNP*DES-generation.Clinically relevant covariates were implicated into following multivariable models.Model D: To answer if DES-generation is associated with death in patients with above median NT-proBNP, death was adjusted by age, sex, smoker, cerebrovascular and/or peripheral vascular disease, and DES-generation.Model E: To clarify no association between DES-generation and death in patients with below median NT-proBNP, death was adjusted by age, sex, DM, CCS class, number of coronary vessels diseased, cerebrovascular and/or peripheral vascular disease, and DES-generation.In order to present the results of model C more clearly, we added two multivariate models.17. To stratify NT-proBNP into two groups of lower and elevated values, the median NT-proBNP of 2927 pg/ml (IQR 1282/8524) was used. As there was an even number of patients in this study, the median of NT-proBNP values was calculated by taking the average of the two middlemost numbers. Contingency tables were used to compare the relative risk of PCI with first- and newer-generation DES for patients with low and elevated NT-proBNP levels. The SPSS 24.0 (IBM Corp.) was used for all statistical analyses16.All variables, which were significant in these COX proportional hazards regression models are shown in Forest plots. With 152 deaths in the whole study population and 110 deaths in patients with NT-proBNP above median, the analyses stick to the rule of thumb with a minimum of ten outcome events per predictor variable. With 42 deaths this target is not exactly met in patients with NT-proBNP below median (seven variables), however, recent reports estimated this rule as too conservative18. Almost half of the patients (n = 164) underwent at least one or more coronary angiographies during the observation period. Table 19. Details regarding cause of death are given in Table This study included 340 patients with CAD and moderate to severe LVSD from the CCLD-MUW. Figure In model A DES-generation was an independent predictor of death in the whole study population including 340 patients (162 first-generation and 178 newer-generation). The baseline characteristics of patients treated with first- and newer-generation DES are presented in Tables Model B demonstrated the distinguished significance of NT-proBNP as a prognostic marker in patients with reduced LVF: NT-proBNP and age were the strongest independent predictors of death. To compare baseline characteristics of patients with different degrees of disease severity we dichotomized the patient population according to the NTproBNP median. The baseline characteristics of patients with NT-proBNP levels below and above the median 2927 pg/ml) are presented in Tables 927 pg/mlModel C showed a significant interaction between DES-generation and NT-proBNP: DES-generation was significantly associated with death, but only in patients with NT-proBNP levels above median.Predictors of death as calculated in different Cox proportional hazards regression models are shown in Table Model D demonstrated an association between death and DES-generation in patients with NT-proBNP levels above median.In model E, which focused on patients with NT-proBNP levels below median, this association could not be confirmed.In order to present the results of model C more clearly, we stratified patients according to their NT-proBNP median and assessed the impact of the DES-generation on the risk of death in different stages of HF: Out of 170 patients with NT-proBNP levels above median 89 patients underwent PCI with first-generation DES and 81 patients underwent PCI with newer-generation DES. Out of 170 patients with NT-proBNP levels below median 73 patients underwent PCI with first-generation DES and 97 patients underwent PCI with newer-generation DES.The relative risk of death in patients who underwent PCI with first-generation DES compared to newer-generation DES was similar in patients with lower NT-proBNP levels (p = 0.558), but was significantly increased in patients with elevated NT-proBNP levels (p = 0.015).Death is associated to stent-generation. NT-proBNP is a predictor for the stent generation used: elevated levels demonstrated a higher mortality risk when using first- compared to newer-generation DES, while lower levels showed a similar risk when using either DES-generation.3. In contrast, our analysis shows, that patients with elevated NT-proBNP levels had a higher mortality risk when using first- compared with newer-generation DES. This finding may result from the fact that heart failure patients are characterized by a prothrombotic activation of the coagulation system, thereby significantly augmenting the risk of stent thrombosis. However, acute coronary events such as stent thrombosis or myocardial infarction are often masked as sudden or pump failure death in these patients.In comparison with first-generation DES, second-generation DES have maintained the low restenosis rates of first-generation devices, but additionally reduced rates of stent thrombosis. However, this difference could not be translated in a survival benefit20 is based on an increased platelet activity as well as elevated levels of blood coagulation markers  and fibrinolytic factors (D-Dimer)20. Recently, a British study reported an association between NT-proBNP and plasma coagulation factors in older men6. In accordance our data clearly demonstrated higher levels of fibrinogen and D-dimer in more severe HF patients defined by higher NT-proBNP levels compared to patients with less severe HF . By hypothetically transforming the autopsy results of the OPTIMAAL trial on our patients additional 5% (8 out of 14 deaths classified as sudden) and 45% (69 out of 85 deaths classified as progressive heart failure) of cases may actually have died from MI, resulting in overall 69% of patients who died from MIIn our analysis Cox proportional hazards regression models were applied to define independent markers of death and to adjust for potential confounders. However, the results of our observational study could be biased by unmeasured confounding. As this study was not a randomized controlled trial patients with first- and newer-generation DES can not be directly compared: the selection of treatment was mainly influenced by time of treatment. It is conceivable that differences in heart failure therapy could have had an impact on the outcome of the two patient groups. However, in the univariate analysis there was no difference in the treatment with neurohumoral antagonists and with implanted cardioverter defibrillators. Accordingly, the results of this analysis have to be interpreted with caution, and are only hypothesis-generating.1. Especially the recently focused bioresorbable stents show thicker stent struts than those of newer generation DES. This should be kept in mind when treating heart failure patients with CAD by PCI. Further studies should evaluate if bioresorbable stents with thicker stent struts are safe in patients with heart failure.In patients with ischemic cardiomyopathy increased NT-proBNP levels predict a higher risk of death after PCI with first- compared to newer-generation DES. Regarding the improved technology of the newer-generation DES, the reduction of the stent strut thickness may be of particular importance, as thicker struts increase the risk of stent-thrombosis


Background: Benzodiazepines (BZD) are common medications for sedative, hypnotic, and anxiolytic that are especially prevalent in older adults. Previous studies have shown that BZD use could impair users' cognition, significantly affecting their quality of life. Past research has shown that higher education might play a protective role in the process of cognitive decline. Very few studies had examined the cognitive effects of BZD on highly educated older adults. The study aimed to explore how cognitive functions would be affected by benzodiazepines among highly educated older adults.Method: 140 older adults with an average education period of 14.8 years were included in this study. The subjects were divided into three separate groups, the long-term BZD users (≥180 days), short-term BZD users (<180 days), and non-users. Demographics and cognitive assessments for the three groups were analyzed using the analysis of variance (ANOVA), the chi-squared test, and the analysis of covariance (ANCOVA). To examine the association between BZD use and cognition a multiple linear aggression approach was used.Result: All three groups were significantly different from each other when looking at executive functioning in the Trail Making Test B (TMT-B). Compared to the control group, short-term BZD users showed significant defects in TMT-B time (p = 0.002) and TMT-B errors (p < 0.001); long-term BZD users showed significant defect on TMT-B time (p = 0.041). Compared to short-term BZD users, long-term BZD users showed significant merit on TMT-B errors (p = 0.001). No significant differences were found in other cognitive tasks that reflected general cognition, verbal memory, language fluency, and visual memory. After adjusting for demographic, increased BZD use over time was positively associated with scores for the revised Brief Visuospatial Memory Test .Conclusion: BZD use may be significantly associated with worse executive functioning in highly educated older adults. However, there is no association between the duration of BZD use and increased cognitive deficits in highly educated older adults. This study identified future experimental directions including potential longitudinal studies, within-subject studies comparing mood disorder patients' cognitive performance before and after onset of BZD use, and between-subject studies that directly compare BZD's effect on subjects with the same baseline of cognitive functioning. Benzodiazepines (BZD) are a class of psychoactive drugs that can enhance the binding of the neurotransmitter gamma-aminobutyric acid (GABA) on the GABA-A receptor, resulting in sedative, hypnotic, and anxiolytic effects . BZDs haIn recent years, several studies have shown that BZD use may impact the cognitive functioning of older adults, but studies varied in the cognitive domains observed and the participant's demographics. A cohort study by Paterniti et al.  using 1,During the process of aging, structural, and functional neurological changes that occur renders older adults more vulnerable to risk factors such as psychotropic drugs, stress, and negative mental health states. However, higher education level is a protective factor of age-related cognitive decline. It has been widely accepted that higher education is consistently related to better cognitive performance in the older adults , 17. TheUnfortunately, very few studies have examined the effect of BZD on cognition, especially for specific cognitive domains, such as memory and executive function, of highly educated older adults. Moreover, the literature currently lacks information on the cognitive effects of BZD in highly educated older adults, and if the duration of BZD use plays a role in cognitive performance. The purpose of this study is to explore if the cognitive function of highly educated older adults is associated with BZD use and how the cognitive effect changes with the duration of BZD use. If higher education level has protective effects on cognitive performance, we expect to see a decrease in lower cognitive performance in the BZD use groups compared to the non-use group. As well, we expect that as BZD use duration increases, there will be no significant decline in cognitive scores as BZD.Participants were recruited from inpatient, outpatient, and annual medical examination services at the First Affiliated Hospital of China Medical University, Shenyang, China from May 2019 to December 2019. This study was approved by the ethics committee in the First Affiliated Hospital of China Medical University. All individuals used in this experiment signed the informed consent.N = 51) were included in the study.The inclusion criteria were (1) aged 60 or older, (2) with a more-than-or-equal-to a high school education level. Participants were excluded if (1) they met the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) diagnostic criteria for Schizophrenia Spectrum and other Psychotic Disorders, and Neurocognitive Disorders, (2) were in a state of anxiety   or depreAfter signing the consent form, participants were evaluated for anxiety and depression symptoms using the SAS and GDS. BZD users are more likely to experience depression and anxiety than the non-users . HoweverN = 47, BZD using time ≥180 day), and short-term BZD users , and non-users  , hypnotic and sedative (N05CD), antiepileptic (N03AE), and myorelaxant (M03BX07). Hypnotic drugs derived or connected with benzodiazepines (N05CF) were also included. Based on the self-report medication history obtained in the interviews and prescription record in the electronic medical record system, we converted the accumulated BZD dosage and average daily BZD dosage of each participant into diazepam equivalents . Based osed BZD) .The cognitive function evaluation process consisted of six cognitive tasks reflecting general cognition and four subdomains of cognition: memory , attention, executive function, and verbal fluency. Cognitive tasks were selected based on high credibility among older adults in China .The Mini-Mental State Examination test (MMSE) (t (MMSE) , 31 is aThe Rey Auditory Verbal Learning Test (AVLT) (t (AVLT) , 33 has The Trail Making Test (TMT) (st (TMT) , 34 is ast (TMT) , 36.The Controlled Oral Word Association Test (COWAT) ( (COWAT) , 38 is aThe Digit Span Test (DST) (st (DST) , 39 is oThe Brief Visuospatial Memory Test-Revised (BVMT-R) ((BVMT-R) , 41 are IBM SPSS Statistics for Windows, Version 22 was used for all analyses. The analysis of variance (ANOVA) tests were used to compare the continuous demographic variables. Chi-square tests were used to compare the classified demographic variables and chronic conditions history of disease among the two BZD use groups and the control group. These statistical tests on the demographic information aimed to identify potential covariates among the three BZD use groups. BZD use information was determined with descriptive statistics. In order to evaluate the cognitive performance among the three BZD use groups, a comparison of cognitive task results was conducted using analysis of covariance (ANCOVA) was performed with BZD use group as a fixed effect adjusting for the demographic variables. Furthermore, to investigate whether BZD use duration can predict cognitive performance, we conducted a bivariate analysis and a multiple linear regression analysis within the long-term and short-term BZD use groups using participants' BZD use duration and cognitive tasks results. The covariates included in the model were age, gender, education, and BMI. A significance level of 0.05 was used for all tests of significance.p = 0.036). Compared to the non-users and short-term BZD users, the BMI of the long-term BZD use group is significantly lower. Among the 93 BZD users, the average accumulated dose of BZD users was 498.13 diazepam equivalent, the average daily dose of BZD users was 0.71 diazepam equivalent, and the average duration of medication was 649.69 days. Seventeen BZD users took BZD because of anxiety and the other 76 participants use it for insomnia.The descriptive demographic information is shown in p = 0.002) and 227.48 ± 113.52 s of long-term BZD users (p = 0.041). As for TMT-B errors, the number of errors of short-term BZD users was 1.85 ± 1.86, which was significant higher compared to both the non-users  and the long-term users .The results of the ANCOVA for differences in cognitive tasks performance with BMI as a covariate is shown in r = 0.235, p = 0.023). After adjusted for demographic variables, the BVMT-R N1 scores of BZD users were still correlated with BZD over time .Consistent with our prediction, multiple linear regression analyses showed no significant decline in the cognitive tests of MMSE, AVLT, TMT, COWAT, and DST with longer duration of BZD use. Interestingly, as shown in In this study, we explored the association between BZD use and the cognitive functioning of highly educated older adults by comparing performance in cognitive tasks for long-term BZD users, short-term BZD users, and non-users. Within our study, the two BZD use groups and the control group were matched in most demographic information, including age, gender, education, smoking status, and drinking status. There was, however, a significant difference in the BMI of the three groups, with BZD user groups having the lowest BMI scores. This is consistent with participants' demographic information in a previous study on BZD use . With thComparing to other studies on BZD use and cognitive functioning, some demographic data of our study were inconsistent. For example, some previous studies included more women in the BZD use group than the non-use group , 6. In oIn the ANCOVA result for differences in cognitive tasks, the three groups only showed significant differences in the completion time and number of errors in the TMT-B test. Previous research has shown that although TMT-B time and TMT-B errors can both reflect participants' executive function, TMT-B time has a stronger correlation with visual movement, while the performance of TMT-B errors has a stronger correlation with working memory , 36. In Although there has been no previous research on the specific cognitive domains affected by BZD use in highly educated older adults, some research on general aged BZD users has reported that the cognitive effects of BZD exist in a variety of cognitive domains including executive function and visual memory , 43–48. In this study, compared to non-users and long-term BZD users, short-term BZD users showed a significant increase in TMT-B errors, reflecting worse working memory function. At the same time, there was no significant difference between the non-users and the long-term BZD users. It was also found that the numbers of TMT-B errors in long-term BZD users were significantly fewer than that of the short-term BZD users, and there was no significant difference between the long-term BZD users and the non-users. In addition, although there was no significant difference in BVMT-R scores among the three groups, there was a significant correlation between the number of BVMT-R N1 scores and the BZD using time among all BZD users. These results suggest that the negative cognitive effects of BZD  in the highly educated older adults did not increase with longer duration of BZD use. Previous studies have shown that long-term BZD use might result in physical dependence , and patPast research on general aged BZD users is consistent with our findings , 46, 50.This study aims to assist with the clinical decision making of prescribing BZD to older adults. The therapeutic effects of BZDs are widely supported, but a major concern with BZD treatment is its potential negative effects on patients' cognitive functions, especially in older patients. With the increase in the accessibility of higher education, as well as the prevalence of BZD prescription, it is expected that there will be an increasing population of highly educated older adults using BZD. This research directly responds to these growing trends, attempting to address an expected clinical question: Are older patients less at risk for the negative cognitive effects BZD treatment's if the patients are highly educated? This forward-seeing perspective of this study provides unique strength and clinical meaning. This study focused on the population of highly educated old adults when investigating the cognitive effect of BZD and controlled for several demographic and clinical variates. Other than BMI, all other demographic information of the groups well-matched.However, there are also some limitations. First, although BZD users were divided into long-term users and short-term users to explore the relationship between the BZD use duration and BZD's cognition effects, as a cross-sectional investigation, this study cannot directly reflect the causal relationship between BZD use and how did the BZD's cognition effects change with using time. Second, due to the outbreak of COVID-19 in China in December 2019, the study had to be discontinued, leaving the sample size of the study unsatisfactory. Third, we did not consider the role of sleep in the cognition effect of BZD. Past research has shown that sleep could influence cognitive performance in older adults  While maThis current study inspires multiple options for future research. The next step of this project could be to design an experimental study that assigns BZD non-users into BZD use and other anxiolytics groups to further reduce the curative effect of the drug on the cognition outcomes. A longitudinal study can also be informative in providing insight into the causality and long-term effects of BZD use. To eliminate the effects of depression and anxiety on cognitive performance, we excluded patients with those conditions. However, because BZD is widely prescribed to manage mood disorders, it is clinically relevant to include those populations in future studies. A preliminary proposal includes a within-subject design examining the cognitive performance of mood disorder patients before and after the onset of BZD treatment. This current study investigated the relationship between BZD use and cognitive performance. Other side-effects of BZD use such as addictive effects should also be evaluated in the context of highly educated adults.In conclusion, we investigated the cognitive effects of BZD use on highly educated older adults and obtained valuable clinical findings. The results of this study found that in the population of highly educated older adults, short-term BZD use is significantly associated worse executive function (TMT), but functioning in general cognition (MMSE) and other cognitive domains such as verbal memory (AVLT), language fluency (COWAT), attention (DST), and visual memory (BVMT-R) was not associated with BZD use. Moreover, the cognition effect of BZD in highly educated old did not increase with the duration of BZD use. The result of this study can contribute to the discourse of the security of BZD use among older adults and provides guidance on clinical medicine in the future.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee in the First Affiliated Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study.LL and YT: conceptualization. LL and MT: data curation and analysis. RZ and YT: project administration. LL, YZ, LJ, and PJ: supervision, writing—review, and editing. LL and PJ: writing—original draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Osteoarthritis (OA) is the most common joint disease and leading cause of pain and disability in the elderly population. Most guidelines recommend the use of non-steroidal anti-inflammatory drugs (NSAIDs) and opioids for the non-operative treatment of OA. Monoclonal nerve growth factor (NGF) antibodies are new drugs with the potential to provide pain relief and functional improvement in OA. We compared the efficacy , and safety of monoclonal NGF antibodies with NSAIDs and opioids in the treatment of OA with a Bayesian network meta-analysis.Results: 38 articles, comprising 41 trials and 20489 patients with OA were included. Overall from the network meta-analysis, anti-NGFs were the most effective drugs for pain relief  and for functional improvement . Although anti-NGFs were associated with higher risk of peripheral sensation abnormality (paresthesia and pruritus), they were not associated with higher risk of other AEs (headaches and nausea) or with higher withdrawal rates related to AEs.Conclusions: Monoclonal NGF antibodies provide significantly greater pain relief and functional improvement in OA compared to NSAIDs and opioids. Monoclonal NGF antibodies are not associated with severe AEs. More studies are needed to confirm these findings.Methods: PubMed, CNKI, Web of Science, Scopus, Embase and Cochrane Library databases were searched for relevant studies  published between January 1999 to January 2020. Bayesian network and conventional meta-analyses were conducted. Pain relief, functional improvement and AEs were assessed. Osteoarthritis (OA) is the most common joint disease. It is the leading cause of pain and disability in the elderly population. It is estimated that at least 300 million people worldwide suffer from OA . OA is aIn the 1950s Levi-Montalcini et al. . ClinicaNumerous systematic reviews and meta-analyses have been conducted to investigate the efficacy and safety of NSAIDs and/or opioids for treatment of OA pain. The goal of our current network meta-analysis was to include the NGF antibodies in this comparison. Based on a recent network meta-analysis that showed NSAIDs and opioids are efficacious in pain relief in OA, we included 13 drugs in our network meta-analysis. These drugs were divided into 5 groups based on activity and mechanism of action: anti-NGFs , potent opioids , weak opioids (tramadol), selective COX-2 inhibitors , and traditional NSAIDs . In a Bayesian network meta-analysis of 41 trials in OA, we assessed drug efficacy, including pain reduction and physical function improvement, and safety.This network meta-analysis was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . A totalA total of 20489 patients were included in the studies. Among the 38 eligible articles, only one study with 385 patients was on hand-joint OA. Across all the trials, the mean age of patients was 61.25 years (range 57.41 to 70.00 years), the percentage of male patients was 32.26% (range 19.57% to 54.03%), and the median follow-up was 84 days (Interquartile range or IQR 42–84 days). The numbers of assessed patients for each treatment were NSAIDs 5408, selective COX-2 inhibitors 4131, anti-NGFs 3108, weak opioids 1405, and potent opioids 1274.The baseline characteristics of the included studies are shown in All the drugs except the potent opioids were superior to placebo for pain relief . The potent opioids had the lowest efficacy and no significant effect  . Based oSubgroup analysis did not reveal any substantial change after excluding the 10 trials that were not commercially funded. Anti-NGFs were still the most efficacious drugs for pain relief  .The anti-NGFs and selective COX-2 inhibitors significantly improved physical function compared to placebo while the potent opioids had no significant effect  . The potThere were significant increases in withdrawal rates related to AEs with anti-NGFs and opioids compared to placebo, but not with selective COX-2 inhibitors  .In the withdrawal related to AEs network, a total of 36 trials were assessed. No significant inconsistency was reported. Significantly higher withdrawal rates related to AEs were reported with potent opioids , weak opioids  and NSAIDs  compared to placebo. Selective COX-2 inhibitors (SUCRA = 93.4%) were the safest, followed by anti-NGFs (SUCRA=53.5%) and NSAIDs (SUCRA=50.5%) . In the A total of 33 trials were selected for assessment. Based on incidence rates, the three most common AEs selected as the secondary safety endpoints were nausea, headache, and peripheral sensation abnormality (paresthesia and pruritus).There were no significant differences in the incidence rates of headache among the three drug classes from the pair-wise meta-analyses. However, potent opioids and weak opioids had significantly higher risks for nausea and peripheral sensation abnormality. In addition, anti-NGFs had significantly higher risk for peripheral sensation abnormality .In the headache network, NSAIDs were the safest drugs . However, there were also no significant differences between the other treatments and placebo. NSAIDs , weak opioids  and potent opioids  had significantly higher risks for nausea. The incidence rate of peripheral sensation abnormality was significantly higher with anti-NGFs , weak opioids  and potent opioids  . No subgselective COX-2 inhibitors were included. All drugs had significantly greater efficacy compared to placebo for both pain relief and function improvement , although overall withdrawal rate related to AEs is not significantly different compared to placebo. Our findings are consistent with the results of previous systematic reviews [The new finding from this study is that monoclonal NGF antibodies have the highest efficacy for pain relief and functional improvement, exceeding that of selective COX-2 inhibitors, NSAIDs, and weak opioids (tramadol), all of which are recommended in the 2019 reviews , 48 and This study has several limitations. Firstly, only randomized, controlled trials (RCTs) were included to avoid uncontrolled confounding factors common in observational studies and other non-RCTs. However, observational studies and other non-RCTs can provide important information on drug efficacy and safety, and omitting these resulted in a small number of studies being included. Secondly, only high-quality studies were included in order to control the quality of the analysis and to minimize the impact of small-sized study effects. However, including small-sized studies can increase generalizability and robustness of the results. Publication bias may be a significant problem, but is difficult to control with a small number of studies. Thirdly, the length of follow-up was not long enough to assess long-term safety outcomes. The median follow-up of studies in this network-analysis of 84 days (IQR 42–84 days) is sufficient to assess AEs that develop relatively quickly but not for long-term safety outcomes such as cardiovascular changes, sensation abnormalities and joint damage. More high-quality RCTs, with long-term follow-up, are needed.A total of 38 studies comprising 20489 patients were included in this network meta-analysis. The results show that monoclonal NGF antibodies provide significantly greater pain relief and functional improvement in OA compared to selective COX-2 inhibitors, NSAIDs, and opioids. Monoclonal NGF antibodies are not associated with severe short-term AEs. More large scale RCTs are needed to confirm these findings.We searched the PubMed, CNKI, Web of Science, Scopus, Embase and Cochrane Library databases, from the start of January 1999 to end of January 2020. Our search terms were 'osteoarthritis', 'tanezumab', 'fulranumab', 'fasinumab', 'oxycodone', 'hydromorphone', 'oxymorphone', 'tramadol', 'celecoxib', 'etoricoxib', 'rofecoxib', 'ibuprofen', 'naproxen', 'diclofenac', and 'paracetamol/acetaminophen'. We also screened the reference lists of relevant systematic reviews and meta-analyses to identify additional eligible articles. All eligible articles were included irrespective of the language of publication.The inclusion criteria for this network meta-analysis were: 1. RCTs; 2. studies comparing the target drugs with placebo, or with each other; 3. studies on patients with OA at any joint; 4. studies reporting the following endpoints: pain reduction, functional improvement, and withdrawal related to AEs. The exclusion criteria were: 1. dose-escalation studies of only one drug; 2. target drugs combined with other drugs; 3. studies for postoperative pain; 4. reviews, systematic reviews and meta-analyses, conference abstracts, letters, pharmacokinetic or pharmacodynamic studies, and studies with insufficient data.For studies with insufficient data, the corresponding authors were contacted to see if the required information could be obtained. If no response was received, a reminder was sent, and if there was still no response the study was excluded. For studies that reported data visually but did not provide numeric values in text or tables, again the corresponding authors were contacted. If no response was received, two authors of the current analysis independently attempted to obtain the data by measurements from the graphs/figures. If that was not possible based on a clear scale and specific reference system then the study was excluded.The Cochrane risk of bias assessment tool was used to perform the methodological quality assessment of the RCTs . The folAuthor, publication year, total sample size, mean age, gender ratio, affected joint, treatments, route of administration, intervention time, follow-up period, and endpoint data were collected and tabulated. To reduce the effect of withdrawal bias, we collected data from the intention-to-treat analysis.The primary efficacy endpoint was pain reduction, and the secondary efficacy endpoint was functional improvement. There were restrictions on the types of questionnaire used in pain evaluation. Functional improvement was evaluated using the function subscale of the Western Ontario and McMaster Universities Arthritis Index (WOMAC). If the WOMAC function score was not measured or reported, the Lequesne Index or other functional measurement scale was used. The change-from-baseline score (mean ± SD) at the last follow-up period was used to evaluate the extent of both pain relief and functional improvement. For studies involving multiple treatment groups with different doses of the same drug, we selected the most effective dose group based on the respective study’s recommendations . We calcSince patient compliance impacts the effect of treatment in clinical practice, we selected the withdrawal rates related to AEs as the primary safety endpoint. The most commonly related AEs were secondary safety endpoints. We calculated the OR with 95% CI for the safety of target drugs versus placebo or versus each other.2 statistic. When there was significant heterogeneity (P<0.05 or I2 >50%), we pooled data and used a random-effects model for analysis. When there was no significant heterogeneity we pooled data and used a fixed-effects model for analysis. The Bayesian network meta-analyses were conducted using Stata/MP  and GeMTC (version 0.14.3). This method increases the number of studies within each comparison and narrows the CI width, resulting in stable results [The efficacies of the target drugs were compared to placebo by direct pairwise meta-analyses using Review Manager Software . Heterogeneity across studies were assessed from the Cochran Q statistic and the I results –54. In t results , 56. The results . Global All estimate outcomes (SMDs or ORs) with 95% CI were generated from the posterior distribution medians. Differences were considered significant if the 95% CI did not include 0 for SMD or 1 for OR. Differences were considered statistically significant at P<0.05. The minimum clinically important difference of -0.37 SD units was selected. This threshold of 0.37 SD units is based on the median minimum clinically important difference reported in studies in patients with osteoarthritis . An effeSupplementary FiguresSupplementary Table 1Supplementary Tables 2-6


In genome-wide association studies, pathway-based analysis has been widely performed to enhance interpretation of single-nucleotide polymorphism association results. We proposed a novel method of hierarchical structural component model (HisCoM) for pathway analysis of common variants  which was used to identify pathways associated with traits. HisCoM-PCA is based on principal component analysis (PCA) for dimensional reduction of single nucleotide polymorphisms in each gene, and the HisCoM for pathway analysis. In this study, we developed a HisCoM-PCA software for the hierarchical pathway analysis of common variants. HisCoM-PCA software has several features. Various principle component scores selection criteria in PCA step can be specified by users who want to summarize common variants at each gene-level by different threshold values. In addition, multiple public pathway databases and customized pathway information can be used to perform pathway analysis. We expect that HisCoM-PCA software will be useful for users to perform powerful pathway analysis. Availability: HisCoM-PCA is available on the website (http://statgen.snu.ac.kr/software/HisCom-PCA/index.html).In genome-wide association studies (GWAS), researchers have identified many single-nucleotide polymorphisms (SNPs) associated with the traits of interest (phenotypes) . HoweverRecently, we presented a hierarchical structural component model (HisCoM) for pathway analysis of common variants (HisCoM-PCA) to identify pathways associated with traits . HisCoM-In our previous study, we used only pathway information of the Kyoto Encyclopedia of Genes and Genome pathway database  and adopThe workflow of the HisCoM-PCA software is shown in The HisCoM-PCA software takes four inputs: (1) a SNP dataset in csv file or PLINK format files, (2) a txt file with phenotype and covariate(s), (3) a set file that consists of two columns for gene name and SNP id, and (4) a set file that consists of two columns for pathway name and gene name. Furthermore, the program also accepts the published pathway databases in MsigDB ,8.The HisCoM-PCA program generates the following output files: (1) a ‘[prefix]. gene-pca_summary.csv’ file that contains the number of SNPs and PCs for each gene, (2) a ‘[prefix].gene.ressum.csv’ file that contains pathway name, gene name, number of permutation, weight of gene for the pathway, gene coefficient, and permutation p-value of gene, and (3) a ’[prefix].pathway.ressum.csv’ file that contains pathway name, number of permutation, number of genes in each pathway, pathway coefficient, and permutation p-value of pathway.We introduced our HisCoM-PCA software for pathway analysis of common variants in GWAS. HisCoM-PCA software supports pathway analyses using multiple candidate pathways and customized PC selection criterion. We expect that our HisCoM-PCA software is useful for users to perform various pathway analyses. This section should contain sufficient detail so that all procedures can be repeated, in conjunction with the cited references. The manufacturer and model number should be stated in this section—for example, as Sigma Chemical Co. .


Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies. Extracellular vesicles (EVs) are small nano-sized particles, secreted by all cell types and capable of encapsulating and transporting several molecules to a target delivery site . EVs canAlterations of the glycosylation pathway are a common feature of malignant cell transformation ,3,4. TheDespite technological advances, the structural characterization of glycans remains quite challenging. The diversity and complexity of these carbohydrates, together with methodological limitations, makes it challenging to deeply analyze the EV glycome ,13. The N-glycans, characterized by an N-linkage to an Asn residue in an Asn-X-Ser/Thr sequon, where X can be any amino acid except proline. N-glycans have a defined core structure, and can be classified depending on their structures and branches in high-mannose, complex or hybrid [O-GalNAc glycans, also known as mucin-type O-glycans, are carbohydrate chains initiated by a GalNAc sugar covalently linked by an oxygen atom to a Ser/Thr residue. They often appear as long ramified structures, and have multiple core structures, the major includes core-1 to core-4 [Glycans are carbohydrate structures that modify both proteins and lipids through a biosynthetic pathway finely regulated by glycosyltransferases and sugar transporters. Glycans can be mainly found at the surface of the cellular membrane forming the commonly known glycocalyx, and they are essential mediators of cell–cell communication and cell–matrix interaction ,14,15. Tr hybrid ; (II) O-o core-4 ; and  [RTKs are a family of receptors that control several cellular signaling pathways involved in numerous biological processes, such as cell proliferation, growth, motility and differentiation . Dysregur (EGFR) ,39,40, hr (EGFR) ,42,43 anr (EGFR) ,45. The r (EGFR) . Additior (EGFR)  and the r (EGFR) ,48, resuN-glycan branching structures have been described to destabilize E-cadherin at the cell surface, and thus promote cell proliferation and tumor progression [Integrins and cadherins are important cell receptor proteins responsible for the interactions between cells and the ECM . Howevergression .x (SLex) and sialyl-Lewisa (SLea), present in either N-glycans, O-glycans or glycolipids. The interaction between selectins and these terminal structures is crucial to the leukocyte “rolling” and extravasation process during inflammation [x and SLea, cancer cells are able to hijack this mechanism to interact with the selectins present in the vascular endothelium, traveling through the circulatory system and thus, potentiate their metastatic behavior [Selectins are another important type of cell adhesion molecule. These include E-selectin, P-selectin and L-selectin which are expressed in the vascular endothelium, platelets and leukocytes, respectively . These lammation . By exprbehavior ,57.+ T cell responses towards tumor cells and causing T-cell anergy. This effect could be reverted through the depletion of galectin [Furthermore, glycans are crucial regulators of the immune response. Aberrant glycosylation can modulate the interaction between tumor cells and selectins, galectins or sialic acid-binding immunoglobulin-type lectins (Siglecs), which are the lectins involved in the immune response (reviewed in ). For ingalectin ,63. An agalectin .N-glycosylation. This is partially a consequence of the increased difficulty of decoding O-glycosylation [N-glycans using PNGase F, there are no reliable enzymatic digestion protocols to isolate O-glycan chains [O-glycans using laborious chemical protocols, such as β-elimination. However, this chemical reaction may induce artifacts due to sample “peeling”, which is a phenomenon characterized by the isomerization and degradation of the innermost monosaccharides from the O-glycan chain [O-glycosylation sites do not have a defined amino acid sequence, in contrast to N-glycosylation. Any Ser/Thr residue in a protein may or may not be modified with an O-glycan chain, making it more difficult to accurately predict O-glycosylation sites [O- and N-glycosylation sites, respectively, based on the provided protein/peptide sequence.In the past years, the field of glycomics has substantially evolved. Nevertheless, this evolution has been slower when compared to other-omics. The fact that glycosylation is not a template-based process, together with its complex structural diversity has slowed the development of tools and methods to accurately characterize glycans structurally ,66. Undosylation . To appln chains ,76. It ian chain . Differean chain . In addion sites ,79. As aO-glycosylation site disclosure, a genetic strategy targeting the COSMC gene, which codifies a key molecular chaperone that together with the C1GalT1 glycosyltransferase is responsible for the O-glycan elongation, was developed [COSMC gene knockout will lead to the shift of the O-glycosylation pathway towards the expression of simple forms of O-glycans, namely Tn and sialyl-Tn. The glycoengineered cell line was named “SimpleCell” (SC). By the targeted purification of the O-glycopeptides and further mass spectrometry characterization, this approach allowed the identification of the protein carriers of these structures and the disclosure of the respective O-glycosylation sites [O-glycoproteins and their O-glycosites with biomarker potential for gastric cancer [To overcome the challenge of eveloped . The COSon sites ,81. For c cancer . More imc cancer .N-glycome and O-glycome of cell lines have been adapted to the study of EV glycosylation. For example, the N-glycome of EVs isolated from glioma [N-glycome of urinary EVs from prostate cancer patients has also been assessed using other high-resolution glycomic methods [O-glycosylation, our group has demonstrated the presence of the tumor-associated antigen, STn by specific antibody recognition in gastric cancer EVs [O-GlcNAcylation, a type of intracellular O-glycosylation, in metastatic colorectal cancer EVs [N- and O-glycosylation profile of EVs from healthy individual urinary samples was also disclosed using high-resolution glycomic techniques, such as matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) [Different high-resolution quantitative mass spectrometry methods previously established to elucidate the m glioma , melanomm glioma  and ovarm glioma  has alre methods . Regardincer EVs . Other gncer EVs . Interesncer EVs . OutsideC/MS-MS) .Given the fundamental impact of aberrant cellular glycosylation in cancer progression and its association with patients’ poor-survival ,4,25,90,EVs are small nano-sized particles that are released into the extracellular space by all types of cells. These vesicles exert a broad array of biological functions, being important mediators of intercellular communication . EV diamEVs are composed of a phospholipid bilayer that provides protection to their cargo against degradation by the proteases and nucleases present in the external environment ,107. EVsCancer EVs are able to mediate communication between cells locally and at a distance, and their cargo can influence the behavior of the recipient cell . ImportaThe unique presence of certain EV surface glycans holds the potential to be targetable for the development of novel EV detection and isolation methodologies. Cancer EVs are rich in high mannose and complex type N-glycans, polylactosamine, as well as sialylated glycans, namely α2,6-linked sialic acids  Figure . DetaileDifferent subpopulations of EVs may share size and biochemical characteristics, which makes it impossible to differentiate or specifically isolate them from a given sample ,126. In Artocarpus integrifolia , that specifically bind to T-antigen, and the Maackia amurensis lectin I (MAL-I), that has a high affinity for Gal or GalNAc residues with an α-linked sialic acid, allowed to isolate different sized urinary EVs from healthy samples based on their surface glycosylation profile, with increased yields and higher purity when compared to CD9/CD81/CD63 antibody-based isolation [Traditional isolation methods are based on EV size and buoyant density, namely, ultracentrifugation-based methods ,133, micsolation . In addisolation . One of solation . Howeversolation . A similsolation . Interessolation . These rsolation . This apsolation .Apart from the affinity-based isolation methods, a commercially available precipitation kit, the ExoGAG, was also developed taking into consideration the glycosylation profile of EVs. This method precipitates EVs out of the solution due to the presence of negatively charged GAG at the EV surface . InteresDespite the technological advances in the field, the study of EVs is still technically challenging. Particularly, innovative and less laborious detection and isolation methods are an urgent need to facilitate an in-depth study of the different EV cargos as a reliable source of biomarkers. The presence of specific patterns of glycans at the EV surface constitute valuable sources to potentiate the development of more sensitive and specific EV detection and isolation methodologies, with the potential to be translated into the clinical setting.Up to date, little is still known regarding the role of EV glycosylation in cancer. Nevertheless, the role and impact of glycosylation in EV biodistribution, uptake and protein cargo sorting have already been proven .N-glycans by PNGase F digestion proved to be more efficient during EV uptake than neuraminidase treatment [It has been described that alterations at the EV terminal sialylation resulted in a re-direction of the EVs to particular organs in mouse models . In thisreatment . In a careatment . It is preatment . In addireatment . For insreatment . Neverthreatment . AltogetConsidering that the presence of certain glycans in cancer cells can regulate the immune response against tumor cells , it is pProteins are sorted into EVs through a variety of mechanisms depending on a multitude of factors, such as the class of EV and the protein to be sorted ,160. ProN-linked glycans and loss of terminal blood group A and B antigens is due to sorting mechanisms of glycoproteins and glycolipids into the EVs [N-glycans [N-glycans in EWI-2 hindered its recruitment to the EVs without altering its whole cellular localization [N-glycan chains, altered the proteome of secreted EVs in prostate cancer [O-glycosylation has also been reported as capable of affecting the sorting of some proteins into EVs, including the CD44 protein [O-glycosylated transmembrane protein has been previously observed to be expressed in EVs carrying truncated O-glycan chains, namely STn [O-glycans that CD44 is increasingly secreted via EVs when compared to the EVs of their normally glycosylated counterparts [N-glycans in an α2,6 position [N-glycosylation site which was speculated to be important in the trafficking of LCN2 into the EV [N-glycosylation, LCN2 was still being sorted into EVs [There has been growing evidence supporting the importance of glycosylation in regulating EV protein sorting. Changes in the glycosylation profile acquired during malignant transformation may interfere with EV protein sorting. It was suggested that the conservation of specific glycan structures, such as increased high mannose, polylactosamine, α-2,6 sialylation and complex -glycans . Both inlization . Recentle cancer . This ste cancer . Further protein . This hemely STn . It was terparts . Interesposition , in EV pposition . This prposition . On the o the EV . Howeverinto EVs . Consideinto EVs ,169.N-glycosylation status of the EMMPRIN, more precisely on the Asn 160 and Asn 268 glycosylation sites [N-glycosylation site at Asn 157, no inhibition of cell adhesion in the recipient cells was observed [Glycans, being part of the EV functional cargo, have been described to play important roles in the cell reprogramming process. EVs derived from breast cancer were able to induce invasion of the recipient cancer cells due to the transfer of the extracellular matrix metalloproteinase inducer (EMMPRIN) . This efon sites . In anotobserved . EVs mayobserved . For insobserved . Taking observed , this stConsidering the critical roles glycans play both in health and cancer situations, further deepening our understanding of the functional roles of EV glycosylation in cancer will open avenues to develop new tools for cancer diagnosis and therapy.EVs are capable of carrying several bioactive molecules and, depending on their surface composition, hold the potential to be used as natural vehicles for localized drug delivery ,174. In EVs can be re-engineered to carry specific molecules, either by manipulating their parental cells or by direct functionalization of the EVs ,176.Several studies have shown the possibility of using glycosylphosphatidylinositol (GPI) as an anchor to attach specific antigens to the membrane of EVs for target therapy purposes. For example, the HER-2 remained stable after its fusion with GPI and incorporation into murine breast cancer EVs, which induced strong HER-2-specific antibody responses when injected into mice . In addiN-desulfated, N-acetylated and glycol-split heparin derivatives [After reaching their target destination, EVs can be incorporated by recipient cells and release their content. As discussed before, the presence of HSPGs on recipient cells has proved to act as receptors for cancer-derived EVs . The strivatives  and a heivatives , that alivatives  or in brivatives .Y in glioblastoma EVs led to an enhanced EV uptake by dendritic cells in a DC-SIGN dependent manner, a receptor involved in the activation of CD8+ and CD4+ T cell responses [Interestingly, it was demonstrated that a specific EV glycosylation coating per se can induce a host immunogenic response. In melanoma, the modification of apoptotic EVs surface towards overexpression of high mannose type glycans, a natural ligand of DC-SIGN, increased the uptake of these EVs by monocyte-derived dendritic cells, leading to an increase in CD8+ T cell response . In addiesponses . TherefoA different strategy with the potential to be applied for cancer therapy involves the hemofiltration of the patient circulating exosomes using the Aethlon ADAPT™ system  . This syThe role of EVs in modulating therapeutic resistance has already been reported (as reviewed in ). EVs caBesides their potential application for cancer therapy, EVs also represent a valuable source of circulating biomarkers . High EVO-glycosylated EVs in pancreatic cancer patients in the early stages of the disease, even when the patient samples were negative for the CA19-9 antigen [Interestingly, the carbohydrate antigen CA125 was identified in serum-derived exosomes from patients with ovarian cancer and the detected levels were significantly higher in the exosomes when compared to the levels detected directly in the serum of these patients . Yokose  antigen . Interes antigen . In addi antigen . In addi antigen .The overexpression or de novo synthesis of particular glycans or glycoconjugates during cancer progression holds the potential to differentiate tumor EVs from benign EVs. Indeed, the proteoglycan glypican-1 (GPC1) and the tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4) were detected in tumor exosomes from heterogeneous samples of pancreatic cancer  or melanO-GlcNAc glycosylation has also been found elevated in breast [O-GlcNAc modification of the transitional endoplasmic reticulum ATPase (TER ATPase) and RuVB-like1 proteins was identified in colorectal metastatic EVs [O-GlcNAc were also detected in TER ATPase as well as in 70 kDa heat-shock protein (HSP70) proteins present in breast cancer EVs, which may act to protect cytosolic and nuclear proteins against degradation [In addition, the n breast  and colon breast , when coatic EVs . Elevateradation . The eleN-glycome of exosomes from hepatocellular carcinoma patient samples was characterized using a reverse capture strategy, and the majority of the N-glycans found in EVs from patients with HCC were modified with sialic acids or fucoses, in contrast to the N-glycans identified in EV from healthy samples [Despite the high potential of EV glycosylation in the discovery of novel cancer biomarkers, studies addressing the glycan profile of blood circulating EVs are still quite scarce. Nevertheless, the  samples . Walker  samples . Interes samples . The eff samples . Althoug samples . In addi samples . NeverthGlycosylation is a post-translational modification that plays a variety of roles in both physiological and pathological contexts . InteresThere is still a large number of questions to be answered regarding the functional role of EV glycosylation in cancer as well as challenges on how to address these questions. Future studies aiming to provide these answers will allow us to discover potential EV glycan-based biomarkers for cancer diagnosis/prognosis/patient stratification and develop innovative therapeutic strategies that can be applied for cancer treatment.