Hearing loss is a modifiable risk factor for dementia in older adults. Whether hearing aid use can delay the onset of cognitive decline is unknown. Participants in this study (aged 62–82 years) were assessed before and 18 months after hearing aid fitting on hearing, cognitive function, speech perception, quality of life, physical activity, loneliness, isolation, mood, and medical health. At baseline, multiple linear regression showed hearing loss and age predicted significantly poorer executive function performance, while tertiary education predicted significantly higher executive function and visual learning performance. At 18 months after hearing aid fitting, speech perception in quiet, self-reported listening disability and quality of life had significantly improved. Group mean scores across the cognitive test battery showed no significant decline, and executive function significantly improved. Reliable Change Index scores also showed either clinically significant improvement or stability in executive function for 97.3% of participants, and for females for working memory, visual attention and visual learning. Relative stability and clinically and statistically significant improvement in cognition were seen in this participant group after 18 months of hearing aid use, suggesting that treatment of hearing loss with hearing aids may delay cognitive decline. Given the small sample size, further follow up is required. By 2050, the number of people affected by dementia is predicted to double to over 131 million ,5,6. HeaA number of individual studies and meta-analyses have reported an association between hearing loss, cognitive decline and dementia, with both peripheral hearing loss and central auditory dysfunction associated with accelerated risks of cognitive decline and incident dementia  were also assessed. Data for other factors likely to influence cognition, such as physical health, social isolation, loneliness, mood, exercise, quality of life and sex were assessed so that their effects on cognition could be considered in the analysis. When ongoing data collection in a healthy aging comparison group of older Australians with typical hearing for their age yields a sample size large enough for meaningful comparison, cognitive and other outcomes will be compared between the hearing aid users and the group who are representative of Australian older adults. The present study investigated the relationship between degree of hearing loss and the extent of cognitive impairment prior to hearing aid fitting. The effect of hearing aid use over time on cognition was also examined, in addition to the effects of hearing aid use on quality of life. This longitudinal study will provide the most rigorous and expansive evidence to date of the effects of hearing aid use on cognition for older adults with hearing loss.This study was carried out in accordance with the recommendations of the Australian National Health and Medical Research Council guidelines for ethical research conduct. The study protocol was approved by the University of Melbourne Behavioural and Social Sciences Human Ethics Sub-Committee (Ethics ID: 1646925). All participants gave written informed consent in accordance with the Declaration of Helsinki.Ninety-nine adults aged 60–84 years, with hearing loss  and no previously diagnosed or suspected cognitive impairment, participated in this study. Seventy-one percent of participants were retired, and 67% had postgraduate tertiary education. Participants had sufficient English to be able to give informed consent, comprehend test instructions and complete questionnaires. The participants were all clients of the University of Melbourne Academic Hearing Aids Clinic and had been identified as suitable to proceed with hearing aid fitting at the time of recruitment. All participants completed a pre-operative (baseline) assessment battery comprising audiometry, speech perception testing, cognitive screening and assessment, and health, quality of life, lifestyle and ease of listening questionnaires. A subset of participants assessed at the 18 month post-operative point completed an identical assessment battery and reported on their hearing aid use.Participants were assessed at baseline by an audiologist in a sound-proof booth, as part of the standard clinical pre-fitting workup. Hearing aids were chosen during a needs discussion with participants based on type and degree of hearing loss, personal aesthetic and technological preferences and communication needs. The NAL-NL2 prescription  was usedScreening for dementia prior to commencing in the study was conducted using the Mini Mental State Examination , a brieCognition in this study was assessed using five subtests from the CogState Cognitive Battery ,32,33,34The Groton Maze Learning Test (GML) assesses executive function, taking 5 min to administer on average. Using a maze learning paradigm, the total number of errors made when attempting to learn the same hidden pathway across five trials presented consecutively is calculated.The Detection Test (DET) assesses psychomotor function and takes 2 min to administer on average. The participant is asked to respond “yes” when a card in the centre of the screen turns over until either 25 correct responses are obtained, or the maximum time limit (2 min) is reached (whichever occurs first). Performance speed (milliseconds) taken to complete the test is recorded.The Identification Test (IDN) assesses visual attention and takes approximately 3 min to administer. A playing card in the center of the screen that turns over is used to create a choice reaction paradigm in which the participant must answer “yes” or “no” to the question “Is the card red?” Performance speed (milliseconds) to complete the test is recorded.The One Card Learning Test (OCL) assesses visual learning and takes on average 5 min to administer. A playing card in the center of the screen that turns over is used to create a pattern separation paradigm in which the participant is required to answer “yes” or “no” to the question “Have you seen this card before in this test?” The test measures performance accuracy.The One Back Test (ONB) assesses working memory and takes 3 min to administer on average. A playing card in the center of the screen turns over to create an n-back paradigm in which the participant must answer “yes” or “no” to the question “Is the previous card the same?” Both speed and accuracy of performance on this task are measured.A detailed health history, including medical history, was taken, , a personal health history, including smoking, current and past alcohol use, illicit drug and medication use.The Hospital Anxiety and Depression Scale (HADS)  measuresHealth status and health-related quality of life (HRQL) for all participants was assessed using the Health Utilities Index-3 quality of life questionnaire , as oneEase of communication in everyday situations and subjective benefit from hearing aids were measured using the Abbreviated Profile of Hearing Aid Benefit , a quesThe International Physical Activity Questionnaire was sued to assess health and lifestyle . The IPThe Lubben Social Network Scale  is a brThe Loneliness Scale  is an 11In evaluating treatment effects, it is important to measure compliance with device use. In addition to using the data logging function of the hearing aids, all participants who were assessed at the 18 month post-fitting point completed a brief questionnaire about their use of their hearing aid/s, including how many hours per day they used it.0 is the average hearing for those with education levels of 12 years or fewer. The slope coefficient β1 is the difference between the averages of the hearing losses for those with 13–15 years education and those with 12 years or fewer. Similarly, the slope coefficient β2 is the difference between the averages of the hearing losses for those with 16 years or more of education and those with 12 years or fewer.Regression was used to quantify differences in hearing loss across education levels. The specification was1 is that an increase of 10 dB in hearing loss in the better ear corresponds to a change of β1 in the mean of Y, controlling for age, gender and years of education.Relationships between cognition, hearing, age, education and sex were quantified at baseline using regression. For a cognition score Y , the regression has the formFor individual participants, clinically important decline in cognition over the study period was identified using the Reliable Change Index (RCI) procedure ,45, wherr = 0.01, p = 0.915). Almost two-thirds (64.6%) of participants had a cardiac condition, with no significant difference in proportions between males and females. Overall, 17.3% of participants had anxiety. Depression was present in only 4.1% of the participants. Loneliness was reported by 43.9% of participants, and 11.2% reported severe loneliness. Only 1 person reported feeling very socially isolated. Of note is that this was a very highly educated sample, with 66.3% of participants with postgraduate education overall. There were 57.1% of the participants who reported carrying out high levels of physical activity according to the IPAQ criterion. The mean HUI3 score for quality of life was 0.74 (quite high relative to the maximum score of 1), with no significant difference between males and females. There were 61.2% of participants who reported no hearing disability according to the HUI3 criterion, 7.1% reported mild hearing disability, 26.5% moderate and 5.1% severe. Baseline characteristics of the study sample are presented in The regression results for Equation (1) in Descriptive statistics for scores across the Cogstate battery are given in r = 0.25), as was greater hearing loss (r = 0.25). Participants with postgraduate education  had significantly better executive function performance than did those with less than 15 years of education. Average cognitive performance across the various medical and demographic factors measured was also calculated, with no differences of note.Pairwise associations of cognitive performance with age, hearing loss and education are shown in (a)An additional 10 dB of hearing loss was associated with an increase in average GML scores by 4.20 points An additional 10 years of age was associated with an increase in average GML scores by 8.16 points , holding the other explanatory variables constant;(c)Having more than 15 years of education was associated with a decrease in average GML scores by 11.26 points  relative to having 12 years or fewer.The statistical interpretations of the coefficients in In addition, the average visual learning (OCL) score was greater by 0.08 points (8.2% of the mean) for those with more than 15 years of education relative to those with 12 years or fewer. The average MMSE scores were significantly better for those with more than 15 years of education, and also for females. Self-reported adaptation and hearing aid usage statistics are shown in p = 0.000). Although the mean bilateral SRT scores also improved after 18 months of hearing aid use, this difference was not statistically significant (p = 0.066).p = 0.001; paired t test). Separate results for males and females suggest that changes of larger magnitude occurred for females.p = 0.028) from the average improvement of 2.95 points for those using their hearing aid/s less than 90% of the time (5.0% of the baseline mean).p = 0.012 for the paired t test), which is also clinically significant. On the hearing disability scale, there were 19 participants who reported increased hearing disability  and 10 who reported reduced hearing disability.Prior to hearing aid fitting, no significant differences in either hearing loss or cognition were observed between males and females in this sample. Although a correlation between hearing loss and age would be expected to be observed in the general population , this waExecutive function (GML) performance was significantly poorer for older participants in this study , and for those with greater hearing loss . The observed decline in mean executive function score with increased hearing loss at baseline cannot be attributed to age since this was controlled in the regression. Executive function was significantly better for participants with greater education  scores by 8% of the overall mean score, as well as significantly improved MMSE scores. As summarized in recent systematic reviews ,14, a nePossible mechanisms underlying the cognitive decline associated with age-related peripheral hearing loss are currently a topic of investigation. A confounding factor of the association is that neuropathic changes and microvascular pathology associated with aging increase the risk of both hearing loss and cognitive decline. In fact, decline in not only hearing but all of our senses is associated with increased cognitive decline . A recenAs previously discussed, a significant limitation of previous research investigating the effects of hearing aid use on cognition is that there has been no evidence of device use or benefit that could be used to examine the longitudinal relationship between the treatment of hearing loss and its effects. In the current study, despite the fact that, on average, the participants had only a mild-to-moderate hearing loss, a significant proportion of these reported becoming frequent users of their aids immediately after fitting (35.3% within a week). Although there was over-reporting regarding device usage when self-report and hearing aid data logging information were compared, data logging information showed that 59% of participants were using their devices at least 60%–90% of the time 18 months after fitting, a far higher than average reported rate of use . There wAt 18 months after hearing aid fitting, group mean scores across the CogState battery showed no significant change, although when the results were separated by sex, mean performance for males on psychomotor function (DET) did decline significantly. A significant improvement in cognitive function across the whole group was observed for executive function (GML), equivalent to 13.2% of the baseline mean score, that would not generally be expected in older adults. Examination of results by sex showed that executive function improved more for females than for males. Reliable Change Index scores also demonstrated either clinically significant improvement or stability in executive function for 97.3% of participants, with decline seen for only one male. Clinically significant improvement was also observed for females for working memory (ONB), visual attention (IDN) and visual learning (OCL).The value of measuring device use was demonstrated in this study, with larger improvements in executive function equivalent to 23.1% of the baseline mean seen for participants who used their hearing aids greater than 90% of the time, whereas participants who used their aids less than 90% of the time improved significantly less (5% of the baseline mean). Females used their hearing aids more regularly and for longer than did males (56.3% vs. 33.3% of the time). A higher prevalence of daily and regular hearing aid use for females has been reported previously ,62, withThe fact that the cohort in this study is highly educated has been noted previously, with significantly better baseline executive function found for participants in this study who had 15 years or more of education. Lower education has been identified as a modifiable risk factor for dementia, with higher cognitive reserve enabling more highly educated people to maintain cognitive function in the presence of brain pathology . Given tAs discussed previously, there are mixed reports in the literature regarding the effects of hearing aid use on cognition in older adults, with some studies reporting a positive effect on cognition ,20,21,22After 18 months of hearing aid use, self-reported overall quality of life was significantly improved. Although it has been shown that generic measures of quality of life do not demonstrate benefits from hearing aid use, the use of disease-specific instruments such as the HUI-3 have been shown to identify positive medium to large effects on quality of life for adults . A diffeAlthough it is common for adults with hearing loss to experience poor mental health, loneliness and isolation ,65,66, tDespite the small sample size to date, both the observed relative stability and clinically and statistically significant improvement in cognition seen in this initial participant group after 18 months of hearing aid use are exciting and encouraging. Of interest also are the observed differences between males and females in hearing aid use and benefit to both quality of life and cognition. If these findings are confirmed with larger numbers in time, this could provide important information for clinical management of older adults of both sexes in the future. Further data collection over time, with increased participant numbers, the addition of genetic screening data and comparative data for a healthy aging ‘control’ group, will confirm whether the treatment of hearing loss with hearing aids can delay or even mitigate against the effects of cognitive decline. Instead of viewing the predicted significant worldwide increase in the incidence of dementia as a looming catastrophe, perhaps the ‘rising tide of dementia’ can be seen instead as ‘both a triumph of public health and an opportunity’ . For the


Cathepsin S, as an adipokine, was reported to play a critical role in various disease, including atherosclerosis and diabetes. The present study aims to elucidate the relationship between circulating cathepsin S and cardiovascular disease (CVD) in patients with type 2 diabetes.A total of 339 type 2 diabetes individuals were enrolled in this cross-sectional community-based study. Basic information, medical and laboratory data were collected. Serum cathepsin S levels were assessed by ELISA.P < 0.001). Moreover, patients with acute coronary syndrome (ACS) had substantially higher levels of serum cathepsin S than those with stable angina pectoris (SAP), with the median 34.65 ng/ml (24.33–42.83) and 25.52 ng/ml (20.53–31.47) respectively (P < 0.01). The spearman correlation analysis showed that circulating cathepsin S was correlated with several cardiovascular risk factors. The univariate and multivariate logistic regression analysis revealed that circulating cathepsin S was an independent risk factor for CVD  after adjustment for potential confounders. Restricted cubic spline analysis showed circulating cathepsin S had a linearity association with CVD. In addition, receiver operating characteristic (ROC) curve analysis demonstrated that the area under curve (AUC) values of cathepsin S was 0.80 , with the optimal cutoff value of cathepsin 26.28 ng/ml.Compared to the CVD (−) group, levels of serum cathepsin S were significantly higher in the CVD (+) group, with the median 23.68 ng/ml (18.54–28.02) and 26.81 ng/ml (21.19–37.69) respectively (Circulating cathepsin S was significantly higher in the CVD (+) group than that in the CVD (−) one among type 2 diabetes. The increased serum cathepsin S levels were associated with increased risks of CVD, even after adjusting for potential confounders. Thus, cathepsin S might be a potential diagnostic biomarker for CVD. Cardiovascular disease (CVD) is the major complication and leading cause of morbidity and mortality in patients with type 2 diabetes . It is pCathepsin S, which belongs to the cysteine proteinase family , is one However, little is known about the direct relationship between circulating cathepsin S and CVD in the context of type 2 diabetes. Hence, we undertook this study and investigated the cross-sectional community-based association between circulating cathepsin S and CVD in patients with type 2 diabetes.This cross-sectional study was conducted in twenty residential areas of Shanghai from February 2004 to July 2004. A total of 339 patients with type 2 diabetes were enrolled. All subjects had written informed consent to participate in the study. Inclusion criteria included established T2DM diagnosis according to the 1999 World Health Organization diagnostic criteria (fasting plasma glucose ≥ 7.0 mmol/L and/or 2 h plasma glucose ≥ 11.1 mmol/L). Exclusion criteria were type 1 diabetes, gestational diabetes, or diabetes induced by steroid use or other endocrine diseases. Based on whether subjects have cardiovascular disease  detected by hospital medical records or a thorough physical examination by trained physicians. These subjects were divided into CVD (−) group (n = 175) and CVD (+) group (n = 164). Moreover, patients with coronary artery disease (CAD) were further sub-divided into stable angina pectoris (SAP) group (n=43) and acute coronary syndrome (ACS) group (n=61) according to their symptoms and clinical examinations. The study protocol was approved by the Institutional Review Broad of Huashan Hospital, Fudan University.A standardized questionnaire was applied by trained physicians to collect basic information, including age, sex, smoking (yes/no), alcohol consumption (yes/no) as well as medical history including diabetes, hypertension and cardiovascular disease. Anthropometric measurements including height, weight, waist circumference, hip circumference, blood pressure, and BMI were taken by trained physicians using standard protocols in duplicate base. Waist circumference was measured with a non-elastic tape held midpoint between the lower rib margin and the top of iliac crest at the end of the expiration phase. Hip circumference was measured as the maximum circumference at the level of the buttocks. Blood pressure was recorded 3 min apart on the non-dominant arm, with an appropriate-sized cuff after the patient had been sitting for 5 min. BMI was calculated as the weight in kilograms divided by the square of height in meters.Peripheral venous blood samples were collected after an overnight fast for at least 10 h. Blood glucose, serum insulin, total cholesterol (TC), triglycerides (TG), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), blood urea nitrogen (BUN), serum creatine and uric acid were measured by using an automatic analyser . Glycated haemoglobin (HbA1c) was determined by high pressure liquid chromatography using an analyser . eGFR was assessed with the CKD-EPI equation . InsulinSerum levels of cathepsin S were measured in duplicate by ELISA , DY1183; R&D Systems, Minneapolis, MN, USA) according to the recommendation by the manufacturer. The intra-assay coefficient of variation was 7.5% to 9.4%, and the inter-assay coefficient of variation was 6.3% to 10.2%.< 0.9), rest pain, as evaluated by echo-Doppler scanning, obstructive disease on femoral angiography, or lower limb revascularization procedure). The presence of CVD was confirmed by reviewing hospital medical records and undertaking a thorough physical examination by trained physicians. Vascular laboratory data, such as electrocardiogram and echo-Doppler scanning of the carotid and lower limb arteries were also performed for all participants. Data on CVD were collected for all the patients.The presence of CVD was evaluated as follows. A detailed medical history was recorded by trained physicians, including previous or current coronary artery disease , cerebrovascular disease , and peripheral vascular disease  were further sub-divided into stable angina pectoris (SAP) group and acute coronary syndrome (ACS) group by symptoms and clinical examinations. SAP was diagnosed as an invariable exertional chest pain lasting 3 to 5 min, and every time, the pain is of the same location, same degree of exertion and excitation provocation, which can be relieved by rest or nitroglycerin. ACS including unstable angina pectoris (UAP) and acute myocardial infarction (AMI). UAP was diagnosed by typical chest pain at rest. And the patients had depressed ST ≥ 0.1 mV, and/or T-wave inversion on electrocardiogram but with normal creatine kinase-MB level. AMI was diagnosed by elevated cardiac biomarkers  and electrocardiogram showed new ischemia (new ST-T change or new left bundle branch block), besides, patients had a prolonged chest pain medical history .In addition, the following definitions were used for cardiovascular risk factors: Hypertension was defined as a systolic blood pressure > 140 mm Hg, a diastolic blood pressure > 90 mm Hg, and/or having received treatment for hypertension. Hyperlipidemia was defined as total cholesterol > 200 mg/dL and/or having received treatment with statins. Hyperglycemia was defined as a serum glucose > 140 mg/dL and/or having received treatment with anti-hyperglycemic medication. The history of smoking or alcohol was defined as never, current , or ever .P< 0.05) was selected as a candidate for the multivariate analysis. Restricted cubic spline (RCS) was constructed to examine the potential relationship between circulating cathepsin S and the risk of CVD incidence. Receiver operating characteristic (ROC) curve analysis was further utilized to check the diagnostic effectiveness of cathepsin S. All statistical analyses were performed using IBM SPSS Version 25 , R version 3.5.2  and Prism 8 software. A two-sided P< 0.05 was considered statistically significant in all analyses.The Kolmogorov-Smirnov test was used to evaluate the normality and variance uniformity of the data. Continuous variables with normal distribution were presented as means ± SD, while variables with a skewed distribution were expressed as median (interquartile range) and log-transformed to approximate normality before analysis. Categorical variables were shown as frequencies and percentages. Differences between groups were examined by Student’s t-tests (for continuous parametric variables), Mann–Whitney U tests (for continuous nonparametric variables) and Chi-squared tests . Spearman correlation was used to estimate the relationship between circulating cathepsin S and other clinical indexes. Univariate and multivariate logistic regression analysis were used to estimate the association between independent variables and CVD (with or without). Variables entered in the multivariate model were chosen according to the clinical and statistical significance. Any variable having significant univariate test  and the mean BMI of 24.76 ± 3.51kg/mP < 0.05). Besides, levels of 2 h insulin (P < 0.001), fasting C peptide (P < 0.05), 2 h C peptide (P < 0.001), TG (P < 0.01), LDL-C (P < 0.01), serum uric acid (P < 0.01) and cathepsin S (P < 0.001) were substantially higher in the CVD (+) group compared with the CVD (−) group. Moreover, patients in the CVD (+) group tended to have higher rates of anti-hypertensive, anti-platelet and lipid lowering drug usages. However, there were no statistical difference in age, sex, smoking status, duration of type 2 diabetes, body mass index (BMI), waist circumference (WC), waist–hip ratio (WHR), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), postprandial blood glucose (PBG), fasting insulin, HOMA-IR, HbA1c, TC, HDL-C, BUN, serum creatine, eGFR and anti-diabetic drug usages between two groups.We divided these patients into CVD (−) group (n = 175) and CVD (+) group (n = 164) based on their diagnosis of CVD. Among CVD (+) group, 44 patients with CAD alone, 30 with cerebral vascular disease alone, 18 with peripheral vascular disease alone, 32 with both CAD and cerebral vascular disease, 20 with both CAD and peripheral vascular disease, 12 with both cerebral vascular disease and peripheral vascular disease, 8 with both CAD, cerebral vascular disease and peripheral vascular disease. All the patients’ clinical and biochemical characteristics are summarized in P< 0.001, As presented in P < 0.01). And patients with ACS had significantly higher levels of BMI (P < 0.05), DBP (P < 0.001), PBG (P < 0.05), 2 h insulin (P < 0.05) and cathepsin S (P < 0.01) than those with SAP. However, there were no statistical difference in other anthropometric parameters, metabolic parameters, and medications between two groups.Since most patients suffered from CAD , we then sub-divided these patients into stable angina pectoris (SAP) group (n=43) and acute coronary syndrome (ACS) group (n=61). As shown in P < 0.05, The Spearman correlation analysis demonstrated that among various metabolic features, cathepsin S was positively correlated with BMI, WHR, SBP, DBP, 2 h insulin, BUN and TG , BMI , PBG , 2 h insulin , LDL-C  and cathepsin S  remained significantly related to the CVD . As shown in To further explore the potential relationship between circulating cathepsin S and CVD, we also used restricted cubic spline (RCS) model. The adjustment factors of RCS analysis for circulating cathepsin S were age, smoking, alcohol consumption and the history of hypertension. Knots of RCS were placed at the 25th, 50th, and 75th percentiles of the serum cathepsin S distribution. Interestingly, we found a significant linear association between circulating cathepsin S and CVD (P < 0.001). And the optimal cutoff value of cathepsin was 26.28 ng/ml, with sensitivity of 62.2% and specificity of 81.1%. These results indicated that cathepsin S level may be a potential diagnostic biomarker for CVD.To evaluate whether cathepsin S could be a diagnostic indicator for CVD, ROC analysis was used to fit the relation. As shown in P = 0.000). Also, we observed that patients with ACS had significantly higher levels of serum cathepsin S than those with SAP. And cathepsin S was associated with several cardiovascular risk factors. In addition, univariate and multivariate logistic regression analysis revealed that circulating cathepsin S was an independent risk factor for CVD . Meanwhile, circulating cathepsin S had a linearity relationship with the risk of CVD. Furthermore, ROC curve analysis showed that cathepsin S may be a potential diagnostic biomarker for CVD with high sensitivity and specificity.In this study, we investigated the relationship between circulating cathepsin S and cardiovascular disease in patients with type 2 diabetes. The major finding was that levels of serum cathepsin S were substantially higher in the CVD (+) group than that in the CVD (−) one  and thereby contributes to the cardiovascular pathogenesis . Active Vascular disease which affects macrovasculature is an important cause of morbidity and mortality in patients with diabetes. A simple and noninvasive tool for predicting and diagnosing CVD in type 2 diabetes patients is of great need. However, numerous studies have only focused on the relationship between cathepsin S and atherosclerosis, cathepsin S and coronary artery disease, cathepsin S and metabolic syndrome, or cathepsin S and diabetes , 32–36, There are several limitations in our study. First, this was a cross-sectional study, so we could not assess the causal relationship between the cathepsin S and CVD, which needs further prospective studies to illustrate. Second, we had a relatively small study population. Although the serum samples were preserved at below −80°C, we cannot preclude the possibility that proteins degrade in some rare cases, which may lead to measurement deviations. In addition, for ethical reasons, we considered it was too problematic to use non-drug-interventions or stop-drug-interventions during follow-up period, so the patients continued to receive anti-hypertensive, anti-platelet, anti-hyperlipidemia and anti-hyperglycemic treatment. Statin medication has been implicated in the lysosomal enzyme activity and release, which may affect serum cathepsin S levels . And it Taken together, the present study found that circulating cathepsin S was significantly higher in the CVD (+) group than that in the CVD (−) one among type 2 diabetes patients. The increased serum cathepsin S levels were associated with increased risks of CVD, even after adjusting for potential confounders. Thus, cathepsin S might be a potential diagnostic biomarker for CVD.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by The Institutional Review Broad of Huashan Hospital, Fudan University. The patients/participants provided their written informed consent to participate in this study.YJ, ZY, and XW designed the study. JS, BL, YY, JW, and RH collected and organized the data. YJ, JS, BL, and WZ did the statistical analysis. YJ wrote the first draft of the paper. BL, WZ, YY, JW, and RH supervised the whole study and gave critical comments to this manuscript. ZY and XW revised the primary manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the grants from the National Natural Science Foundation of China , the Science and Technology Commission of Shanghai Municipality .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.


The course of Alzheimer’s disease (AD) varies between individuals, and the relationship between cognitive and functional decline and the deterioration of behavioral and psychological symptoms of dementia (BPSD) is still poorly understood. Until recently, it was challenging to monitor subsequent changes in these symptoms because there was no single composite scale available that could simultaneously evaluate activities of daily living (ADL), BPSD, and cognitive function (CF) states. The present authors developed a new, brief assessment scale, the “ABC Dementia Scale” (ABC-DS), which is based on item response theory and facilitates concurrent measurement of ADL, BPSD, and CF states. We previously presented the reliability, construct validity, concurrent validity, and responsiveness of the ABC-DS. We obtained the evidence through three clinical trials featuring 1,400 subjects in total. In the present study, we performed a secondary analysis of the data obtained in the previous study. We conducted hierarchical cluster analyses that allowed us to classify 197 AD patients in terms of similarities regarding ADL, BPSD, and CF domain scores, as measured by the ABC-DS. Consequently, the scale identified subgroups of patients with global clinical dementia ratings of 1, 2, and 3. Considering our results in conjunction with the clinical experiences of the AD expert among the present authors regarding longitudinal changes in ADL, BPSD, and CF, we were able to propose potential progression pathways of AD in the form of a hypothetical roadmap. Patients with Alzheimer’s disease (AD) are very heterogeneous regarding their profiles of activities of daily living (ADL), their behavioral and psychological symptoms of dementia (BPSD), and their cognitive function (CF).1 - 42, total tau, and phosphorylated tau [Previous researchers classified subtypes of AD using various methods such as clinical subtypes defined by diagnostic criteria ; atrophyated tau ; and subated tau  and prinated tau . Stagingated tau . IdentifTo effectively treat AD, clinicians must understand the precise progression pathways of the disease, considering changes in ADL, BPSD, and CF. For this purpose, we need a conceptual roadmap that indicates the paths of the changes in ADL, BPSD, and CF that patients are likely to experience during disease progression. By presenting these pathways, such a roadmap would contain much more information than the ratings from the currently available assessment scales for dementia.For the present study, we used PubMed to find numerous studies that examined longitudinal changes in AD symptoms ; howeverWe have previously validated a brief assessment scale—the “ABC Dementia Scale” (ABC-DS)—which can concurrently evaluate ADL, BPSD, and CF in ten minutes on average . We deveIn a recent study, we used hierarchical cluster analysis classify individuals with a global Clinical Dementia Rating (CDR)  of 0.5 iThis study involves a secondary analysis of data obtained in the TRIAD1412 clinical trial . For TRITo determine the severity of AD, trained clinical psychologists rated the 312 patients in terms of CDR. The numbers of patients with a CDR score of 0, 0.5, 1, 2, and 3 were consequently determined to be 4, 110, 99, 65, and 33, respectively; one subject was unavailable for rating. The clinical psychologists used the Japanese version of the CDR and participated in a training seminar to ensure the standardization of testing. For the secondary analysis in this article, we extracted patient data with CDR of 1, 2, and 3; the total sample size was 197 out of 312.http://www.umin.ac.jp/, No. UMIN000021134).We conducted the TRIAD1412 study following the requirements of the World Medical Association’s Declaration of Helsinki (1964) and its amendments and subsequent clarifications . The insWe developed the assessment scale and used the item response theory to examine the quality of the questions. We repeated this process twice  and obtained a candidate version for a confirmation study. We validated the candidate by statistical approaches in TRIAD1412 . FinallyThe ABC-DS is a composite scale to evaluate the levels of ADL, BPSD, and CF. The scale comprises 13 questions (Q1–13), and each item (question) is answered using a nine-point level, ranging from 1 to 9, or from worst to best. Evaluators interview caregivers regarding recent episodes within one month concerning their patients and choose the most suitable score for each item. No specialized training is required to perform the interviews, and the evaluation time was 10 minutes on average for TRIAD1412 (data not shown).We originally developed this scale for clinical trials so that we could compare the effects of different treatments. To measure the overall severity of dementia and the states of ADL, BPSD, and CF, we extensively revised the questionnaire descriptions by inspecting the quality of the items. We performed this revision by investigating item response category characteristic curves, following the item response theory . We statWe can use the ABC-DS to evaluate the effectiveness of medical interventions by comparing patients’ scores before and after the intervention. There are four approaches for such an evaluation: count per item, domain score, the sum of the 13 items , and TDD score. The domain scores for ADL, BPSD, and CF are calculated by summing the following item scores: “Q1 + Q2 + Q3 + Q4 + Q11 + Q12,” “Q7 + Q8 + Q9,” and “Q5 + Q6 + Q10 + Q13,” respectively. We estimated overall severity using total score or TDD score defined byAlthough the total score can detect changes in the overall severity, it has a disadvantage; if a patient’s score on the ADL domain increases by 2 points between assessments but the score on the CF domain decreases by 2 points, after adding the two scores to obtain the total score, these changes cancel out to zero. Thus, pathological changes would remain undetected between two measurements. However, TDD can identify the difference in the score. Therefore, we used TDD scores to estimate the overall severity in this study.Our previous research on the concurrent validity of ABC-DS with standard assessment scales  found thabc_scale@tri-kobe.org) for any commercial use and clinical trials.Researchers can download the ABC-DS questionnaire in English, French, Chinese, and Korean from the following site under the terms and conditions specified: https://eprovide.mapi-trust.org/instruments/abc-dementia-scale. Any amendment in the ABC-DS questionnaire is not allowed. As TDD is a patent-protected technique, approval is required from the owner  to evaluate the precision and repeatability of the repeated measures.stats and hclust (https://www.rdocumentation.org/packages/fastcluster/versions/1.1.25/topics/ hclust), respectively, for hierarchical cluster analysis.Using the three-domain scores  of the ABC-DS, we performed hierarchical cluster analysis and used three clustering options “complete,” “average,” and “Ward” methods in hclust . BecauseWe compared the ABC-DS with other accessible assessment scales  in terms of 12-week responsiveness . If the We cut the cluster dendrogram “tree” at the first generation (branch) and classified the patients with baseline CDR scores of 1, 2, and 3 into four, five, and three subgroups, respectively . We thenWe characterized each subgroup along with GI, GII, and GIII by comparing them with C1.1, which served as a reference. For GI, the BSPD score was almost unchanged, but CF scores decreased. For C1.3, the ADL, BPSD, and CF scores declined. The differences between C.1.1 and GII were characterized by decreases in ADL and CF scores, with a slight deterioration in BPSD. The BPSD levels of C2.4 and C3.2 were comparable to that of C1.1, but the ADL and CF scores were considerably lower. GIII represents the advanced stage of the disease with high severity and showed markedly lower ADL, BPSD, and CF scores when compared with C1.1. C3.3 represents the terminal stage.The TDD score of the ABC-DS detected the changes in scores over 12 weeks for individuals with a baseline CDR of 0 and 0.5, 1, and 2. However, analysis of the total score for all 13 items in the ABC-DS only indicated changes among individuals with a baseline CDR of 0/0.5. MMSE and CDR-SOB failed to detect differences when the baseline CDR was 0/0.5. Using the ABC-DS, we could precisely monitor the dynamic changes in ADL, BPSD, and CF for AD patients.Through cluster analyses, we classified 197 AD patients and found subgroups for patients with baseline CDR scores of 1, 2, and 3. Because these subgroups probably captured stages in progression, we hypothetically constructed the progression pathways by reasonably placing these subgroups in a roadmap, as shown in To interpret the roadmap, we classified the arrow indicators with the following three kinds: blue, which indicates changes without deterioration in BPSD; red, which indicates deterioration of ADL, BPSD, and CF; and others, which mainly show a worsening of BPSD.We interpreted the roadmap as follows. Patients moving from C1.1 to GI do not have BPSD. Patients at GI mainly take one of two pathways in terms of progression: GI to C2.4, in which ADL and CF deteriorate, but BPSD is not present; and GI to GII, in which BPSD appears and ADL and CF levels decrease. There is also a possibility to progress from GI to C1.3. Patients who move from C1.1 to C1.3 or GI to C1.3 could have BPSD at the early stage of progression. Patients who move from GI to C2.4 and C.3.2 could fail to show deterioration in BPSD to a great extent in their clinical course; however, patients at C2.4 may progress to GIII, showing apparent deterioration in BPSD. The patients in GII progress to GIII through a decline in ADL, BPSD, and CF. Patients from C1.3 to GIII show a decrease in ADL and CF levels. We define C3.3 as the terminal stage, which is dominated by patients from GIII. BPSD may not be evident in patients at C2.4 and C3.2, and some patients may take a pathway to the terminal stage, but the others at C2.4 or C3.2 could remain in the present subgroups.We searched for relevant literature in PubMed to interpret the pathways in the road map as follows. Feldman reported that an MMSE score of 16 appears to be a turning point at which most instrumental ADL was lost, and the significant losses in basic ADL began to occur over the following 12 months . As an MLiu-Seifert et al. suggested that functional impairment is primarily driven by and follows the cognitive decline in mild AD dementia . They liFinally, Barocco et al. failed to prove, through an examination over a four-year time interval, the existence of the so-called “fast decliners” regarding AD progression . NotablyOur study had limitations because our results of cluster analyses were preliminary and cross-sectional in nature; they represented only a static snapshot of the dynamically changing AD symptoms. The frequency of the paths (i.e., the arrows in The ABC-DS classified AD patients into subgroups characterized by ADL, BPSD, and CF states. In a previous study, we reported that the ABC-DS also identified subgroups in individuals considered to have probable MCI. Accordingly, we have determined that the ABC-DS can precisely monitor dynamic changes in the symptoms of individuals with conditions ranging from probable MCI to severe AD. To build upon our findings, we must confirm the roadmap using longitudinal data, showing changes from probable MCI to severe AD.For the accurate estimation of overall severities, TDD is better than total scores because the total score neglects the fact the ABC-DS is composed of three dimensions.Click here for additional data file.


The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline  was applied intravitreally in rats ( Glaucoma, besides age-related macular degeneration and diabetic retinopathy, ranges among the most common causes for irreversible vision loss worldwide. These numbers will further increase by 2030 . GlaucomN-methyl-d-aspartate (NMDA)-induced excitotoxicity model, an ocular hypertension glaucoma model (OHT), and a model of chronic hypoperfusion through bilateral occlusion of the carotids. In all models, an increased expression of HSP27 at the protein and mRNA level could be detected in the retina [HSPs are ubiquitously expressed, have a highly conserved structure, and are induced in response to various physiologic and environmental stressors. They perform chaperone functions by stabilizing new proteins and helping to refold proteins that were damaged by different types of stress . HSP27 ie retina . In the e retina ,11. Addie retina . For thie retina ,19. On oe retina . Furthere retina . Yokoyame retina . On the e retina . In preve retina ,25. In ae retina . HSP27 ie retina . DependiIn this study, we investigate the direct effects of an intravitreal HSP27 injection on the retina and the optic nerve. Here, we noted a degeneration of RGCs, amacrine cells, and the optic nerve neurofilament after HSP27 injection. We assume that the high, non-phosphorylated extracellular level of HSP27 activated cell death mechanisms.p > 0.09, Intraocular pressure (IOP) was measured weekly before and for three weeks after the injection A. No dif2  in the a-wave amplitude  recordings after 21 days. No significant changes were found in the amplitude of a-wave C and b-wmplitude C.p > 0.9; Haematoxylin and eosin (HE)stained retinas were evaluated A. The in+ cells demonstrated a decrease in the in the RGC number in HSP27 group (65.5 ± 11.4%) compared to the PBS group  compared to the PBS group  and the control group  and the amount of activated microglia/macrophages (ED1+ and Iba1+) were investigated  compared to controls  Iba1+ signals were counted in the GCL, 35.4 ± 17.3% (p = 0.79) in the IPL, and 15.6 ± 2.4% (p = 0.53) in the INL. No significant differences between the groups could be detected in these layers  microglia/macrophages were observed in the PBS (20.1 ± 2.9%) and the HSP27 group , while lower cell counts were detected in the IPL  and INL . Again, no significant difference between the two groups could be detected in these cell layers  and the PBS group  could be detected regarding the LFB score (Myelin sheaths of optic nerves were stained with luxol fast blue (LFB) 21 days after the intravitreal injection A. No difFB score B. Hence,p = 0.049; However, neurofilament damage was detected. Nerves were stained with anti-SMI-32 antibody to visualize possible disruptions of the axonal neurofilaments A. In con+ microglia/macrophages in optic nerves of the HSP27 group (125.2 ± 11.7%) and the PBS control group  did not differ between the investigated groups  [At day 21, the thickness of the retina was unaltered and its general structure remained intact, while we found a significant loss of RGCs. These results are in accordance with previous studies, where a systemic immunization with HSP27 induced RGC loss after five weeks ,25. RGCsd (NMDA) ,33,34. Id (NMDA) ,35. In ad (NMDA) . Hence, d (NMDA) ,38,39. Id (NMDA) . Also, td (NMDA) . In mousd (NMDA) . NF-κB cd (NMDA) .+ T-cell response [Another theory for the degeneration is the involvement of the immune system. Previously, in sera of glaucoma patients, an elevated antibody titer against HSP27 was detected . Wax et response . This in+ bipolar cells. Only recoverin+ cone bipolar cells were affected [2, pointing towards a small damage to signal transmission of the photoreceptor cells. This was also observed for 1 cd.s/m2 after the intravitreal injection of S100B [To examine the degeneration more closely, the other neurons of inner retinal layers were also examined, more specifically amacrine and bipolar cells. Both are interneurons of the retina and important for visual signal transfer, since they modulate and transfer signals to the RGCs . We noteaffected . In a glaffected . In our of S100B . Chien eof S100B . Perhapsof S100B .In order to investigate the effect of intravitreally injected HSP27 on the optic nerve, the neurofilament and the myelin sheaths were analyzed. A degeneration of the neurofilament was noted, while the myelin sheaths remained unaffected. Since we observed retinal degeneration, axonal degeneration in the present study is possibly the result of direct injury to the cell bodies of the RGCs. This type of degeneration is also called Wallerian-like degeneration. Wallerian-like degeneration  was alsoSince a degeneration in retina and optic nerve was observed, we also analyzed glia cells. HSP27 acts intracellularly as a chaperone and is generally considered to be protective . In contAstrocytes are the most common glial cells in the optic nerve head and can enter the retina. After glaucomatous damage, astrocytes strongly react in the retina and the optic nerve of humans . GFAP, tAnimal experiments and animal care procedures adhered to the ARVO Statement for Use of Animals in Research and the animal care committee of North Rhine-Westphalia . In this study, 24 male Wistar rats  were involved. All animals had unlimited access to food and water and were kept on a light-dark cycle (12 h:12 h). Detailed health checks and eye exams were performed regularly.n = 12) was injected with 2 µL of 0.2 µg/µL  HSP27 solution under a stereomicroscope , with a 32-gauge needle . Control animals received the same volume of PBS , since this was used as a solvent for HSP27. After the injection, the eyes were treated with an antibiotic ointment . No injection was performed on the contralateral eye.The intravitreal injections of HSP27 or PBS were performed as previously described . Brieflyn = 6/group) was measured using a rebound tonometer  one day before the intraocular injection (baseline). IOP was also recorded 7, 14, and 21 days after the injection (The IOP of both groups (njection A. Means n = 6/group) were performed as previously described [2 were recorded. Signals obtained from the corneal surface were thereafter amplified, digitized, averaged and stored using ERG View 4.380R software . In this case, a low pass filter (150 Hz) was necessary. Amplitudes of a-waves (response of photoreceptor cells) and b-waves (response of cells in the inner nuclear layer) were exported to Excel  and then Statistica  for further evaluation.The ERG measurements (escribed . Before n = 6/group) and optic nerves (n = 6/group) were explanted and prepared for histological cross-sections or longitudinal sections, respectively. Eyes were fixed for one hour in 4% paraformaldehyde , whereas optic nerves were fixed for two hours. Afterwards, the tissue was cryo-conserved in 30% sucrose overnight and frozen embedded in NEG-50 Tissue Tek medium . Retinal cross-sections (10 µm) and longitudinal optic nerve sections (4 µm) were mounted on glass slides . Thereafter, the cuts were fixed in ice-cold acetone for 10 min.After 21 days, eyes  were obtained. The retina was dissected from the eye bulb and then stored at −80 °C before being processed.n = 6/group) were done via HE staining (both Merck). Retinal pictures were taken at 200× magnification  and retinal thickness was measured from the ganglion cell layer to the outer nuclear layer by using the tool “line” from Zen 2012 software . The thickness of the retina was measured three times per retinal image. A total of 12 values were used to calculate the mean value .Structural analyses of retinas . Three pictures per LFB stained optic nerve section  were taken at 400× magnification. The LFB stained optic nerves were scored from 0 = intact myelin up to 2 = strong demyelination, in 0.5 intervals [The myelin alterations in the optic nerve were examined using LFB staining  and six longitudinal optic nerve sections (n = 6 nerves/group) were blocked with a mixture of a 10–20% serum, 0.1–0.2% Triton X-100 , and PBS . Primary antibodies  were prepared as previously described [For western blot analysis, retinas (escribed . Brieflyt-test and is presented as mean ± standard error of the mean (SEM). A p < 0.05 was considered statistically significant . All results of the PBS control group were set to 100% and compared to the HSP27 group.The data were analyzed using Statistica  using a Student’s In summary, we noted an IOP-independent loss of RGCs and amacrine cells accompanied by a neurofilament degeneration after intravitreal HSP27 injection. At 21 days, glia cells were not affected. However, it could be too late for a microglia/macrophage response and too early for a macroglia response. Hence, high extracellular HSP27 level caused a neuronal degeneration. As glaucoma patients also have an increased level of HSP27 in the retina, this is of particular interest. Our results might be helpful to understand the role of HSP27 during glaucoma. However, the exact pathomechanisms, leading to the observed neurodegeneration, should be investigated further.


Intravitreal EDN1 was sufficient to drive RGC death in vivo. EDN1 insult caused JUN activation in RGCs, and deletion of Jun from the neural retina attenuated RGC death after EDN insult. However, deletion of Ddit3 did not confer significant protection to RGCs after EDN1 insult. These results indicate that EDN caused RGC death via a JUN-dependent mechanism. In addition, EDN signaling is known to elicit potent vasoconstriction. JUN signaling was shown to drive neuronal death after ischemic insult. Therefore, the effects of intravitreal EDN1 on retinal vessel diameter and hypoxia were explored. Intravitreal EDN1 caused transient retinal vasoconstriction and regions of RGC and Müller glia hypoxia. Thus, it remains a possibility that EDN elicits a hypoxic insult to RGCs, causing apoptosis via JNK-JUN signaling. The importance of EDN-induced vasoconstriction and hypoxia in causing RGC death after EDN insult and in models of glaucoma requires further investigation.Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs), the output neurons of the retina. Multiple lines of evidence show the endothelin  system is important in glaucomatous neurodegeneration. To date, the molecular mechanisms within RGCs driving EDN-induced RGC death have not been clarified. The pro-apoptotic transcription factor JUN  and the endoplasmic reticulum stress effector and transcription factor DNA damage inducible transcript 3  have been shown to act downstream of EDN receptors. Previous studies demonstrated that JUN and DDIT3 were important regulators of RGC death after glaucoma-relevant injures. Here, we characterized EDN insult in vivo and investigated the role of JUN and DDIT3 in EDN-induced RGC death. To accomplish this, EDN1 ligand was intravitreally injected into the eyes of wildtype, Six3-cre There are three secreted EDN peptide ligands  and two EDN membrane-bound receptors (EDNRA and EDNRB). EDN ligands are the most potent vasoactive peptides known, and the canonical role of EDN signaling is to mediate vasoconstriction8. Changes in blood flow have been documented in human12 and animal models6 of glaucoma, and it is hypothesized that these changes could be important factors in the development and progression of glaucoma. EDN ligands and receptors were found to be upregulated in models of acute14 and chronic ocular hypertension17. Importantly, molecular clustering analysis of transcriptional changes in ocular hypertensive mouse retinas and optic nerve heads revealed that upregulation of Edn ligands and receptors was among the first molecular changes in glaucoma pathology6. Levels of EDN were also found to be elevated in the aqueous humor and plasma of glaucoma patients20. Antagonism or knockout of EDN receptors lessened RGC loss in models of acute and chronic ocular hypertension5. In addition, EDN delivery was sufficient to drive RGC death in vivo21. Despite these findings, the cell death pathways responsible for eliciting RGC death upon EDN insult remain unknown. Understanding the molecular mechanisms controlling EDN-induced RGC death in glaucoma will provide insight into early, critical pathways of glaucomatous neurodegeneration and can identify potential therapeutic targets for neuroprotective glaucoma treatments.Glaucoma is a neurodegenerative condition affecting the output neurons of the retina—the retinal ganglion cells (RGCs). The mechanisms that cause RGC insult and death in glaucoma remain incompletely understood. Recently, the endothelin  system has been implicated in glaucomatous neurodegeneration26. JNK signaling is a mitogen activated protein kinase phosphorelay system where MAP3Ks phosphorylate and activate MAP2Ks (MKK4 and MKK7), which phosphorylate and activate MAPKs (JNK). JNK phosphorylates and activates its canonical target JUN, which can then act as a proapoptotic transcription factor by promoting the transcription of pro-death genes 28. Jun deficiency or loss of function was significantly protective to RGCs after chronic ocular hypertension25 and axonal injury26. Similarly, endoplasmic reticulum stress has been implicated in neurodegenerative diseases, including glaucoma29. Prolonged endoplasmic reticulum stress promotes translation of DNA-damage-inducible transcript 3 , which can act as a pro-apoptotic transcription factor. Ddit3 deficiency was moderately protective to RGCs after axonal injury and ocular hypertension30. Dual deletion of Jun and Ddit3 conferred additive, near complete, and long-term protection to RGCs following optic nerve crush26. Given the potential importance of EDN signaling in glaucomatous neurodegeneration, it is plausible that EDN causes RGC death via JUN and/or DDIT3 activation. Notably, both JUN and DDIT3 have been known to act downstream of EDN receptors. MAPKs (including JNK) and JUN were shown to be upregulated or activated in a variety of cell types, including cultured RGCs, after exposure to EDN40. Therefore, JUN may play a role in regulating RGC death after EDN insult. The EDN system has also been shown to activate the endoplasmic reticulum stress response42. Therefore, it is possible that JUN and/or DDIT3 are RGC-intrinsic regulators of EDN-induced RGC death, similar to after glaucoma-relevant injuries. Here, we characterized the time course and extent of EDN insult and critically test the importance of JUN and DDIT3 in RGC death following EDN insult in vivo.JUN N-terminal kinase (JNK) signaling has been shown to be important in mediating glaucoma-relevant neurodegeneration44. Thinner retinal vessels and evidence of hypoxia have been demonstrated in animal models of chronic glaucoma46. Importantly, JUN was found be an important regulator of ischemia-induced neuronal death51. Thus, it is possible that EDN elicits a hypoxic insult to the retina via EDNRA to ultimately cause JUN-dependent RGC loss. To clarify how EDN ligands may be acting to cause RGC loss, we characterized changes in retinal vessel diameter and hypoxia after EDN insult.In addition to the apoptotic RGC-autonomous mechanisms triggered by EDN signaling, the cell types that respond to EDN ligands to ultimately cause RGC death remain unknown. EDN ligands cause vasoconstriction by binding to EDNRA expressed by vascular smooth muscle cellsDdit352  or floxed alleles of Jun53 (Junfl) were utilized for all experiments. All mice used were 2–6 months of age. Floxed alleles were recombined in the optic cup using Six3-cre54 . All alleles were backcrossed >10 times to the C57BL/6J genetic background (>99% C57BL/6J), and all colonies were maintained by intercrossing. Mice were fed chow and water ad libitum and housed on a 12-h light-to-dark cycle. Roughly equal numbers of males and females were used for each experimental group. Animals were randomly assigned to experimental groups. Before experiments were performed, it was established that animals with pre-existing abnormal eye phenotypes  would be excluded from the study. All experiments were conducted in adherence with the Association for Research in Vision and Ophthalmology’s statement on the use of animals in ophthalmic and vision research and were approved by the University of Rochester’s University Committee on Animal Resources.Wild-type (WT) C57BL/6J mice and mice carrying a germline deletion of Mice were anaesthetized with an intraperitoneal injection of 0.05 ml/10 g solution containing ketamine (20 mg/ mL) and xylazine (2 mg/ mL). Eyes were sterilized with 50% betadine solution made in 1X phosphate buffered saline (PBS), and were thoroughly flushed with 1X PBS. The conjunctiva at the temporal quadrant was cleared away with the bevel of a 30-gauge needle, and a small incision was made with the 30-gauge needle through the sclera and behind the limbus. Vitreous was allowed to drain, and any blood was wicked away. Five μL Hamilton syringes  with a blunt 33-gauge needle were used to perform intravitreal injections. The needle of the Hamilton syringe was inserted 1 mm into the incision site at a 45° angle toward the optic nerve, and was held in place for 30 s prior to injection. Care was taken to avoid contacting the lens with the Hamilton needle. Before experiments were performed, it was established that eyes with observable lens damage as a result of intravitreal injection would be excluded from the study. Over the course of 2 min (to prevent a sudden increase in intraocular pressure), 2 μL of 500 μM EDN1 , diluted in sterile 1X PBS was injected into the vitreous. The Hamilton needle was held in place for 30 s following the injection, and the needle was removed from the eye over the course of 30 s. Antibiotic ointment was placed over the eye. The contralateral eye was injected with 2 μL of vehicle (sterile 1X PBS) with identical methods to serve as a control. Note: the investigator was masked to genotype while performing intravitreal injections.55. Mice were anaesthetized with an intraperitoneal injection of 0.05 mL/10 g solution containing ketamine (20 mg/mL) and xylazine (2 mg/mL). Mice were given an intraperitoneal injection of 0.2 μL/g fluorescein (25% Fluorescein Sodium) immediately prior to angiography. Retinal vasculature was photographed using a Micron III mouse retinal imaging system (Phoenix Research Labs).Fluorescein angiography was performed as previously describedHypoxia was assessed using pimonidazole HCl (Hypoxyprobe) according to manufacturer’s instructions. Ninety minutes prior to euthanasia, mice were given an intraperitoneal injection of (60 mg/kg) pimonidazole HCl diluted in sterile 1X PBS. Mice were then transcardially perfused with 20 mL saline solution and 20 mL 4% paraformaldehyde. Eyes were harvested and incubated in 4% paraformaldehyde for 30 min. Eyes were stored in 1X PBS until dissection. Hypoxia was detected using an antibody against pimonidazole  overnight, 20% sucrose overnight, and 30% sucrose for 3 days. Eyes were embedded in tissue freezing medium , frozen at −80 °C, and cross-sectioned at 14 μm. Sections were collected on glass microscope slides, which were stored at −20 °C. For hypoxia detection, one section every 84 μm was collected for immunofluorescence to ensure representation from the entire retina. Sections were blocked in 10% horse serum, 0.1% TritonX in 1X PBS for 3 h at room temperature and then incubated in primary antibody made in 10% horse serum, 0.1% TritonX overnight at 4 °C. Sections were washed in 1X PBS and incubated in secondary antibody made in 1X PBS for 2 h at room temperature. Sections were then washed, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and mounted in Fluorogel in TRIS buffer (Electron Microscopy Sciences). A masked observer captured images. For hypoxyprobe immunofluorescence, images were exposure-matched.56. All sections analyzed were a maximum of 168 μm from the optic nerve head. Five equidistant 20× fields (444 μm) were captured per section; two at each periphery, two at each middle area, and one at the center. Four sections were analyzed per retina. The cell counter tool in ImageJ was used for cell quantification. For each section, cell counts were summed. The score of each retina was the average of the 4 sections’ summed cell counts normalized to the distance measured.Methodology for cell quantification in retinal sections was adapted from Harder et al.For whole mount staining, retinas were dissected free from the optic cup and blocked in 10% horse serum, 0.4% TritonX in 1X PBS overnight at 4 °C. Retinas were incubated at 4 °C for 72 h in primary antibody diluted in 10% horse serum, 0.4% TritonX in 1X PBS. Retinas were then washed and incubated overnight at 4 °C in secondary antibody diluted in 1X PBS. Retinas were washed and mounted ganglion cell layer-up in Flourogel in TRIS buffer (Electron Microscopy Sciences). Note: for triple immunofluorescence, primary antibodies from three different host species were utilized, and secondary antibodies conjugated with flourophores with 3 different excitation/ emission frequencies were utilized.26, RBPMS+, SOX2+, and pJUN+ cells were quantified using eight 40× fields per retina, and cCASP3+RBPMS+ cells were quantified using eight 20× fields per retina. Images were equally spaced 220 μm from the peripheral edge of the retina. The cell-counter tool in ImageJ was used for quantification. A masked observer captured and quantified images.As previously described57.To assess changes in artery and vein diameter after EDN1 insult ex vivo, mice were trancardially perfused with 20 mL PBS and 20 mL 4% PFA 5 min after EDN1 injection. Retinas were processed for whole mount immunostaining as described above. Retinal artery and vein diameters were measured using the line segment measurement tool in ImageJ. Six measurements per CD31+ artery and vein were taken 240 μm, 305 μm, 370 μm, 435 μm, 500 μm, and 565 μm from the optic disc. Distance from the optic disc was chosen in order to include measurments before the approximate location of the first arterial bifurcations. Arteries and veins were identified by their distinct branching morphologies58. Mice were anesthetized with an intraperitoneal injection of 0.05 ml/10 g solution containing ketamine (20 mg/mL) and xylazine (2 mg/mL). Meloxicam (2 mg/kg) was administered subcutaneously prior to surgery. The optic nerve was exposed and crushed immediately behind the eye with self-closing forceps for 5 s. Antibiotic ointment was applied to the eyes after the procedure.Controlled optic nerve crush (CONC) was performed as previously described2, and SOX2+ Müller glia/mm after PBS and EDN1 in WT mice; Figs. F test to compare variance and a Shapiro–Wilk test to test normality to ensure appropriate statistical tests were utilized. %RGC survival .Data were analyzed using GraphPad Prism8 software. Power calculations were performed before experiments were conducted to determine appropriate sample size. Data from experiments designed to test differences between two groups  C57BL/6J mice were intravitreally injected with 2 μL of 500 μM EDN1 diluted in 1X PBS (1 nmol EDN1). 1X PBS was intravitreally injected into the contralateral eye to serve as an internal vehicle control. Note, this injection paradigm was used for all experiments. In models of chronic ocular hypertension, RGCs die via apoptosis58, a process mediated by cleavage of caspase 3. Therefore, retinas were immunoassayed for activated caspase 3  at 3, 5, 10, and 14 days post-EDN1 and PBS injection. Retinas were counterstained for RBPMS, an RGC-specific marker59, to ensure caspase 3 activation was specific to RGCs. Intravitreal EDN1 caused caspase 3 activation in RBPMS+ RGCs; this activation peaked 5 days post injection and was sparse by 14 days post injection. cCASP3+ RGCs were rarely present at any timepoint post-PBS injection  JUN and RBPMS 1 day following PBS or EDN1 injection  of cCASP3+ RBPMS+ cells were also JUN+  and WT  controls were intravitreally injected with EDN1 or PBS, and retinas were assessed for apoptotic RGCs and subsequent RGC loss. Jun deletion significantly lessened caspase 3 activation in RGCs 5 days post insult  is important in mediating EDN1-induced RGC death. DDIT3 , a pro-apoptotic transcription factor activated after prolonged endoplasmic reticulum stress, moderately contributed to RGC death after glaucoma-relevant insults60. Importantly, previous studies have demonstrated that JUN and DDIT3 additively contributed to RGC death after axonal injury 26. Therefore, we tested the effect of Ddit3 deletion alone and in combination with Jun deletion on RGC survival after EDN insult. To accomplish this, mice with a homozygous germline deletion of Ddit3 (Ddit3−/−) and WT littermate controls were utilized. Ddit3 deletion did not significantly reduce caspase 3 activation 5 days post-EDN1 compared to WT controls, and Ddit3 deletion did not significantly prevent RGC death 28 days post-EDN insult. To determine whether DDIT3 and JUN act in tandem to drive RGC death after EDN insult, Ddit3−/−Jun−/− eyes were intravitreally injected with EDN1 or PBS. Dual deletion of Ddit3 and Jun was significantly protective to RGCs 28 days post injection, but this was not significantly different than the effect of Jun deletion alone. Therefore, EDN1-induced RGC loss was largely JUN-dependent.Notably, 44. Importantly, JUN-JNK signaling has been shown to mediate neuronal death after ischemic insult51. Therefore, it is possible that EDN1 elicits a hypoxic insult to the retina, which can ultimately lead to RGC death via JNK-JUN signaling. To determine whether EDN1 elicited retinal vasoconstriction, fluorescein angiography was utilized at several timepoints post-intravitreal injection of either EDN1 or PBS. As previously reported in rats63 and rabbits65, intravitreal EDN1 caused rapid retinal vasoconstriction, which lasted at least 3 h post injection. Retinal vessels appeared normally reperfused after 6 h. No vascular changes occurred at any timepoint post-PBS  was utilized to determine whether intravitreal EDN1 caused retinal hypoxia. Pimonidazole forms covalent adducts in cells with a partial pressure of oxygen <10 mmHg. EDN1-injured retinas had hypoxic RBPMS+ RGCs and Müller glia somas (labeled by SOX2) and processes beginning 3 h post injection (data not shown) and lasting at least out to 24 h  immunostaining was utilized to assess loss of amacrine cells and disruption of synaptic strata in the inner plexiform layer after EDN1 insult. After 28 days, EDN1 did not cause loss of ChAT+ amacrine cells (in the inner nuclear layer or the ganglion cell layer) or appear to disrupt the synaptic layers of the inner plexiform layer 26 and chronic ocular hypertension (47% protection)25. In contrast, we found Ddit3 deletion did not provide significant protection from EDN-induced RGC death. These data do not preclude the possibility that Ddit3 deletion protected a small portion of RGCs after EDN insult that was not detected with our experimental design. This possibility is not unlikely, as Ddit3 deletion protected only 25% of RGCs 120 days post-axonal insult26 and 20% of RGCs in ocular hypertensive mice30. Nevertheless, these data suggest that DDIT3 did not govern the majority of RGC death following EDN insult. Future work should elucidate the upstream regulators of JUN activation and the downstream targets of JUN signaling to further clarify the molecular mechanisms governing RGC apoptosis in response to EDN signaling. For instance, MAP3K12 (DLK)26, MAP2Ks 4 and 7 79, and MAPKs 9 and 10 22 are upstream regulators of JUN activation and were shown to be important in mediating glaucoma-relevant RGC death, and are perhaps important in regulating EDN-induced RGC death.In the present study, JUN was activated in 30% of RGCs 1 day post-EDN1 Fig. , and Junoss Fig. . This prJun deletion protects RGC axons as well as somas after EDN insult. JUN has been shown to regulate RGC somal degeneration, but not axonal degeneration, after RGC axonal injury and chronic ocular hypertension. Theoretically, if EDN1 injures RGCs at the level of the soma, Jun deletion would likely confer protection to RGC axons as well as somas. However, if EDN1 causes a primary injury to RGC axons, Jun deletion could potentially confer protection to RGC somas, but not axons. Further dissecting the role of Jun in the degeneration of RGC somas and axons will be important for understanding EDN1-induced RGC death, and can possibly classify the location of EDN1 insult to RGCs .Furthermore, it will be important to determine whether In addition to the cell death pathways important in eliciting RGC death after EDN insult, the retinal cell types that respond to EDN ligand and ultimately cause RGC death remain unknown. A multitude of cell types express EDN receptors; retinal neurons (including RGCs) and macroglia are known to express EDNRB, while RGCs and retinal mural cells are known to express EDNRA. While it is possible that EDN ligands affect RGCs directly (via receptors expressed by RGCs), it is also possible that EDN ligands signal extrinsically to RGCs by affecting glia or vascular cells and eliciting neurotoxic downstream effects; ultimately causing JUN-mediated RGC death. Regardless of the primary cell type EDN1 acts upon to ultimately cause RGC death, the present study indicates that ultimately, JUN is important in RGC degeneration after EDN1 exposure.43. EDN ligands bind to EDNRA expressed by vascular mural cells, which elicits contraction of vascular smooth muscle cells and consequential vasoconstriction44. Thinner retinal vessels and reduced blood flow have been reported in human80 and animal models of glaucoma6. Notably, JNK-JUN signaling was found to contribute to neuronal death after ischemic insult to the rodent brain50 and retina51. Therefore, it is possible that aberrant EDN signaling via mural cell-EDNRA contributes to reduced retinal blood flow in glaucoma, which could in turn perpetuate neurodegeneration via JNK-JUN signaling.The canonical role of EDN signaling is to mediate vasoconstriction63 and rabbits65, we found intravitreal EDN1 caused potent but transient arterial constriction in the mouse retina, which reperfused by 6 h post injection (Fig. 46. RGC death did not necessarily occur in close proximity to major vessels after EDN1 insult, but it is possible that loss of blood flow or damage to minor vessels and capillary beds contributes to EDN pathology. Future studies should investigate the effect of EDN on retinal vessel integrity, and how any changes may play a role in EDN-induced RGC death. Severe hypoxia is known to cause RGC and amacrine cell loss, however, we observed RGC loss (Fig. Consistent with previous reports using ratsion Fig. . Subsequion Fig. . Hypoxicoss Fig.  but not oss Fig. ; consist


APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy. The DNA cytosine deaminases APOBEC3A and APOBEC3B have emerged from cancer genomics studies as drivers of mutation in cancers and tumor heterogeneity. Here the authors present a computational approach to identify the RNA mutations specifically driven by APOBEC3A, and developed an RNA mutation-based assay to quantify ongoing APOBEC3A activity in tumor cells. Members of the APOBEC3 family are capable of deaminating cytosine into uracil in DNA and/or RNA4. In particular, APOBEC3A (A3A) and APOBEC3B (A3B), which target the TpC motif in single-stranded DNA (ssDNA)7, are shown to be the main source of APOBEC-signature mutations in patients’ tumors3. A3A and A3B are typically expressed after viral infection to defend cells against foreign DNA or RNA, but are not expressed in proliferating normal cells4. However, A3A and A3B expression is detected in ~50% of tumors9. In breast, lung, head & neck, esophageal, bladder, and cervical cancers, A3A and A3B are abnormally expressed, which is associated with high levels of APOBEC-signature mutations10. The mutagenesis induced by A3A and A3B in cancer cells may not only contribute to tumor evolution but also confer a vulnerability. The A3A and A3B activities in cancer cells induce DNA replication stress and DNA breaks13, offering an opportunity to target the cancer cells harboring high levels of A3A/B activities by disrupting the DNA damage response (DDR)14. To exploit the A3A/B-induced vulnerability of cancer cells, it is critical to quantitatively measure the A3A/B activities in tumors. In this study, we investigated how to specifically quantify the activity of A3A in tumors.Recent cancer genomics studies on tumors of a variety of cancer types have revealed important mechanisms driving tumor evolution and presented opportunities for cancer therapy. Among the 30+ mutational signatures identified to date, at least two are associated with APOBEC  proteins15. In addition, we recently found that A3A but not A3B displays a preference for substrate sites in DNA stem-loops8. Importantly, recurrent APOBEC-signature mutations in different cancer patients are not randomly distributed in the genome but enriched in DNA stem-loops, suggesting that A3A is a major driver of mutations in tumors. Similar to its preference for DNA stem-loops, A3A also recognizes RNA stem-loops in a sequence-specific manner16. The distinct sequence and structural preferences of A3A and A3B have made it possible to distinguish the contributions of these enzymes to mutagenesis in tumors.While A3A and A3B have similar cytidine deaminase activities, their substrate specificities are not identical. Using yeast as a model to characterize A3A/B-mediated mutagenesis, the Gordenin laboratory identified differences at the substrate sites of A3A and A3B, with A3A preferring the YTCA motif and A3B favoring the RTCA motif (R is a purine and Y is a pyrimidine)14. Importantly, A3A-expressing cells become dependent on the Ataxia Telangiectasia and Rad3-related protein (ATR) checkpoint pathway to tolerate the DNA damage caused by A3A expression14. Inhibition of ATR14 or its effector kinase Chk114 leads to increased cell death in an A3A-dependent manner, suggesting the potential use of ATR or Chk1 inhibitors to target tumors with high A3A activity. Furthermore, suppression of translesion synthesis (TLS) or base excision repair (BER), which are involved in tolerating and removing uracil in DNA, strongly sensitizes A3A-expressing cancer cells to ATR inhibitors10. We previously showed that depletion of endogenous A3A and A3B from certain cancer cell lines reduced their susceptibility to ATR inhibition, suggesting that both A3A and A3B may contribute to the vulnerability of tumors.In addition to their differences in substrate specificity, A3A and A3B are also distinct in their catalytic activities. A3A is much more catalytically active than A3B, but A3B is typically expressed at higher levels than A3A in tumors. When overexpressed in cancer cells, A3A induces DNA replication stress, DNA double-strand breaks (DSBs), and cell cycle arrest in a catalytic activity-dependent manner17, raising the possibility that persistent high A3A/B expression is not tolerated in cancer cells. Finally, due to the low expression of A3A, both A3A protein and A3A mRNA are difficult to quantify in limited numbers of cancer cells, making it particularly challenging to predict the levels of currently ongoing A3A activity in tumors.The quantification of A3A/B activities in tumors has been a challenge to date. Cell-extract-based in vitro assays have been developed to measure the activities of A3A/B. Although these in vitro assays are instrumental for understanding the biochemical properties of A3A/B, they are not easily applicable to tumor samples because of the large number of cells required. Furthermore, although given enough time A3A and A3B lay down clear mutational footprints in the genome, these footprints do not directly correlate with the currently ongoing activities of the enzymes. Interestingly, a recent study suggested that APOBEC mutagenesis occurs in an episodic manner in tumorsIn this study, we developed a strategy to quantify the ongoing activity of A3A in tumors. We found that A3A expression and A3A-mediated DNA mutagenesis in tumors, but not those of A3B, correlate with APOBEC-signature mutations in RNA stem-loops. These RNA mutations are not present in their DNA templates, suggesting that they are directly generated by A3A. Using hotspot APOBEC-signature mutations in RNA stem-loops identified from A3A-positive tumors and droplet digital PCR (ddPCR), we developed a quantitative and sensitive assay to measure the RNA-editing activity of A3A. Because of the labile, transient nature of RNA, the RNA-editing activity of A3A accurately reflects the currently ongoing activity of A3A. We demonstrate that the RNA mutation-based A3A assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the currently ongoing A3A activity on DNA. Finally, we show that the RNA mutation-based A3A assay can be applied to clinical samples from cancer patients. Together, our results present a new strategy to follow the dysregulation of A3A in tumors, which will provide new opportunities to investigate the role of A3A in tumor evolution and to target A3A-induced vulnerabilities in cancer therapy.18. To identify the APOBEC+ tumors dominated by A3A or A3B, we sought to separate the contributions of A3A and A3B to the APOBEC-signature mutations  oligo containing a single TpC site is incubated with cell extracts. When the C in the TpC site is deaminated by the APOBECs in cell extracts, the resulting U is cleaved by uracil DNA glycosylase (UNG), leading to an abasic site (AP site). The AP site in the ssDNA then undergoes site-specific breakage in alkaline buffer at 95 °C, which can be visualized and quantified by electrophoresis  is a strong substrate of A3A in vitro  is a quantitative and sensitive method to detect SNPs, rare mutations, copy number variations, and gene rearrangements. ddPCR is based on the partitioning of PCR reactions into small droplets following the Poisson distribution and resulting in partitions containing zero, one or more copies of the template sequence. After PCR amplification, the droplets are individually assessed for fluorescence, allowing an absolute quantification of the template sequence. Importantly, two fluorescence probes can be used simultaneously to determine the proportions of two sequence variants in a population of molecules. Given these desirable features of ddPCR, we tested whether it can be used to quantify A3A-generated RNA mutations in cells.WT) or a catalytically inactive A3A mutant (A3AE72A) upon doxycycline (DOX) treatment . Significant RNA-editing activity was detected in NCI-H2347 and BICR6, two cell lines expressing A3A   for the reference gene, which allow us to compare the A3A mRNA levels in these samples by qPCR  cellsPCR Fig. . Two of RNA Fig. . Thus, w4. APOBEC-mediated mutagenesis in cancer cells may contribute to tumor evolution in several ways. The mutations generated by APOBECs may activate oncogenes and inactivate tumor suppressors, increasing the proliferative or survival advantages of cancer cells22. APOBEC-induced mutations may also promote tumor heterogeneity, increasing the ability of tumors to metastasize or develop resistance to therapy21. Furthermore, we and others recently showed that A3A/B expression can induce replication stress and DNA damage, generating genomic instability beyond mutations14. The genomic instability induced by A3A/B offers an opportunity for targeted therapy. For example, A3A expression in cancer cell lines confers sensitivity to ATR inhibitors14. To understand the role of A3A in tumor evolution and to target the A3A-induced vulnerabilities of cancer cells, it is critical to develop a quantitative and sensitive assay for measuring A3A activity in tumors.From recent cancer genomics studies, APOBEC proteins have emerged as key drivers of mutagenesis in a variety of cancers17. It is possible that A3A is transiently activated by viral infection or other oncogenic stresses in cancer cells, leaving mutational footprints even after A3A is no longer expressed. It is also possible that persistent A3A expression is not well tolerated in tumors because of its robust activity and its ability to induce high levels of genomic instability, thereby selecting for tumor cells that turn off A3A after transient expression. Regardless of the mechanism underlying the separation of A3A footprints and expression, our findings suggest that the A3A mutational signature is not a reliable predictor of currently ongoing A3A activity in tumors.Using the distinct mutational signatures of A3A and A3B, we developed a computational strategy to identify the tumors dominated by A3A or A3B. Surprisingly, in the tumors dominated by A3A, the levels of A3A-signature mutations do not correlate well with A3A mRNA levels, suggesting that A3A mutational footprints cannot reliably predict currently ongoing A3A activity in tumors. Consistent with our finding, a recent study suggested that APOBEC-signature mutations are generated in an episodic manner in tumors24, suggesting that A3A may recognize RNA substrates in a slightly different way. Our finding showed the activity of A3A on RNA closely correlates with its activity on DNA, suggesting that the current impact of A3A on DNA can be monitored by following A3A-mediated RNA editing. Nonetheless, the specific activities of A3A on equivalent DNA and RNA substrates may not be identical.A3A is known to modify not only DNA but also RNA in response to viral infection. In this study, we found that A3A-signature mutations are detected in RNA from tumors even in the absence of corresponding DNA mutations, suggesting that these RNA mutations are directly generated by A3A. The RNA mutations correlate with the expression of A3A but not A3B in tumors. Similar to the DNA mutations generated by A3A, the RNA mutations induced by A3A are enriched in stem-loops. However, in RNA stem-loops, the sequence surrounding the UpC motif differs from the sequence surrounding the TpC motif in DNA stem-loops. For example, in RNA stem-loops with 4-nt loops, the UpC motif is most often preceded by A or U, whereas in DNA stem-loops, the TpC motif is usually preceded by C or T. We speculate that the difference of sequence preference is necessary to compensate for the structural difference between RNA and DNA backbones. The structure of A3A bound to single-stranded DNA showed several direct interactions between A3A and the sugar-phosphate backbone of DNAC) in loops of four nucleotides, many A3A-generated RNA mutations in tumors convert one Isoleucine codon to another Isoleucine codon (AUC > AUU). In tumors, only a few missense mutations were detected in the RNA edited by A3A. Whether some missense RNA mutations generate gain-of-function or dominant-negative protein mutants remains to be investigated. The possible impacts of RNA mutations on non-coding RNAs also need to be explored. The fraction of edited RNA for each RNA target in tumors is typically 1–10% averaging across patients  has recently approved the use of ddPCR for monitoring the response of chronic myeloid leukemia (CML) patients to tyrosine kinase inhibitor treatment26. In future studies, it will be critical to test whether the RNA mutation-based A3A assay is sensitive enough to detect A3A activity in different types of tumor samples from cancer patients.In this study, we demonstrate that the RNA mutation-based A3A assay can be applied to blood samples from blood cancer patients. Even in low-quantity samples that cannot be reliably analyzed by A3A RT-qPCR, the RNA mutation-based ddPCR assay detected A3A activity. It should be noted that ddPCR is already being used to detect pathogens, viruses, copy number variations, and rare mutations14. Inhibition of ATR in A3A-expressing cancer cells leads to replication catastrophe and cell death, and suppression of TLS or BER further enhances this sensitivity to ATR inhibitors10. This dependence of A3A-expressing cancer cells on the ATR checkpoint offers an opportunity for targeted therapy using ATR inhibitors. A number of clinical trials of ATR inhibitors are already underway28. The RNA mutation-based A3A assay that we developed may enable identification of tumors harboring significant A3A activity, helping to identify patients who may benefit from ATR inhibitor therapy. We envision that additional therapeutic strategies will be developed to exploit the A3A-induced vulnerability of cancer cells. The RNA mutation-based A3A assay may also be applied to facilitate these future therapies. In addition to guiding therapy, the RNA mutation-based A3A assay can be used to monitor the dysregulation of A3A during tumorigenesis. These studies may help us understand when, why and how A3A is activated and inactivated during tumor evolution. Defining the contexts and the windows of action for A3A may help address whether and how A3A contributes to tumor heterogeneity, metastasis, and drug resistance. We anticipate that the RNA mutation-based A3A assay will significantly advance our understanding of the function of A3A in tumorigenesis and allow us to exploit A3A-induced vulnerabilities in cancer therapy more effectively.Cancer cells with significant A3A activity rely on DNA repair pathways and the ATR checkpoint to tolerate A3A-induced replication stress and genomic instabilityPaired tumor-normal sequencing data from The Cancer Genome Atlas (TCGA) and other projects was analyzed to study molecular correlates of APOBEC enzyme activity at the DNA and RNA levels. Data from whole-exome DNA sequencing (WXS), whole-genome DNA sequencing (WGS), and RNA sequencing (RNA-Seq), was included in the analysis, all aligned to human genome build hg19. Detailed descriptions of the computational analysis are available in APOBEC3A and APOBEC3B cDNA were synthesized by GenScript with a beta-globin intron and a Flag tag at C-terminus. The catalytically dead mutant APOBEC3A-E72A was constructed by site-directed mutagenesis. The plasmid expressing APOBEC3A-Flag, APOBEC3A-E72A-Flag, and APOBEC3B-Flag were generated by inserting the cDNA into pInducer20 or pDEST53 vectors using the Gateway Cloning System (Thermo Fisher Scientific).−1)10. U2OS derivative cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. For proteins expression, U2OS cells were incubated with doxycycline (200 ng mL−1) for 48 h. U2OS, RPE1-hTERT, and HEK-293T cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. OVCAR5, PC9, and NCI-H2347 cells were maintained in RPMI-1640 GlutaMAX-I supplemented with 10% FBS, 1% penicillin/streptomycin, 1% glucose, and 1% sodium pyruvate. TOV21G, OV17R, and BICR6 cells were maintained in DMEM/F12 GlutaMAX-I supplemented with 10% FBS and 1% penicillin/streptomycin. SKBR3 was maintained in McCoy’s 5A supplemented with 10% FBS and 1% penicillin/streptomycin. The cell lines above were purchased from either ATCC or Sigma-Aldrich.U2OS-derived cell lines were generated by infecting U2OS cells with lentivirus expressing APOBEC3A, APOBEC3A-E72A, or APOBEC3B under a doxycycline-inducible promoter (pInducer20) and selected with G418 (400 μg mLsiRNA transfections were performed by reverse transfection with Lipofectamine RNAiMax (Thermo Fisher Scientific). siRNAs were purchased from Thermo Fisher Scientific (Silencer® Select siRNA). Cells were treated with indicated drugs 16 h after siRNA transfection. The sequences of the siRNAs used in this study were:siCTL: Catalog #4390846siAPOBEC3A: CGACAGUACCAGACUCCAUttsiAPOBEC3B: CCUCAGUACCACGCAGAAAttThe antibodies used in this study were: Flag-M2 monoclonal antibody (Sigma-Aldrich #F1804)GAPDH polyclonal antibody (EMD Millipore #ABS16), Flag polyclonal antibody (Sigma-Aldrich #F7425), APOBEC3B monoclonal antibody (Abcam #ab184990), APOBEC3A/B polyclonal antibody (Santa Cruz #sc-86289), and Ku70 monoclonal antibody (Gene Tex #GTX70271).Gemcitabine was purchased from Selleckchem and dissolved in DMSO. Doxycycline and purified human Interferon-αA/D was purchased from Sigma-Aldrich.10. Cells were lysed in 25 mM HEPES (pH 7.9), 10% glycerol, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM MgCl2, RNase A (0.2 μg ml−1) and 1 mM ZnCl2, and protease inhibitors. Cell lysates were sonicated, incubated for 30 min at 4 °C and then centrifuged 10 min at 16,000 × g at 4 °C. Protein concentration of the supernatant was determined by Bradford assay (Bio-Rad).The APOBEC deamination assays were performed with cell extracts from the indicated cell lines10. Reactions (50 μL) containing 8 μL of a normalized amount of cell extracts (expressing A3A or A3B) were incubated at 37 °C during 1 h in a reaction buffer (42 μL) containing a DNA oligonucleotide , 1.5 units of uracil DNA glycosylase (NEB), RNase A (0.1 μg mL−1) and 10 mM EDTA). Then, 100 nM of NaOH was added to the reaction following by 40 min at 95 °C. Finally 50 μL of formamide was added to the reaction  and the reaction was incubated at 95 °C for 10 min following by 5 min at 4 °C. DNA cleavage was monitored on a 20% denaturing acrylamide gel  and run at 65 °C for 80 min at 150 V. DNA oligonucleotide probes were synthetized by Thermo Fisher Scientific. The sequences of DNA oligonucleotide probes used in this study are:The deamination assays were performed as previously describedTCGGGAAAAAAA-3′PolyA-TC: 5′-(6-FAM)-AAAAAAAAATCAGCTTGCTGA-3′NUP93: 5′-(6-FAM)-GCAAGCTGTTotal RNA was extracted from cells using RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Following extraction, total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT products were analyzed by real-time qPCR using SYBR Green  in a CFX Connect Real-Time PCR detection system (Bio-Rad). For each sample tested, the levels of indicated mRNA were normalized to the levels of Actin mRNA. The sequences of the PCR primers used in this study are:Actin-forward: CCAACCGCGAGAAGATGAActin-reverse: 5′-CCAGAGGCGTACAGGGATAG9APOBEC3A-forward: GAGAAGGGACAAGCACATGG9APOBEC3A-reverse: TGGATCCATCAAGTGTCTGG9APOBEC3B-forward: GACCCTTTGGTCCTTCGAC9APOBEC3B-reverse: GCACAGCCCCAGGAGAAGDDOST558C>T amplification) or 40 ng (for CYPIF13222C>T amplification) of cDNA, and indicated primers (2 μL) were added in the PCR reactions (ddPCR Supermix for Probes (No dUTP) mix from Bio-Rad) in a total of 25 μL. Then, 20 μL of the reaction mix was added to a DG8 cartridge (Bio-Rad), together with 70 μL Droplet Generation Oil for Probes (Bio-Rad) following by the generation of droplets using a QX200 Droplet Generator (Bio-Rad). Droplets were next transferred to a 96-well plate before to start the PCR reaction in thermal cycler  under the following conditions: 5 min at 95 °C, 40 cycles of 94 °C for 30 s, 53 °C for 1 min, and then 98 °C for 10 min (ramp rate: 2 °C s−1). Droplets were analyzed with the QX200 Droplet Reader (Bio-Rad) for fluorescent measurement of fluorescein amidite (FAM) and hexachloro-fluorescein (HEX) probes. Gating was performed based on positive and negative DNA oligonucleotide controls. The ddPCR data were analyzed with QuantaSoft analysis software (Bio-Rad) to obtain fractional abundances of edited RNAs. Three or more biological replicates were analyzed for each sample. DDOST and CYFIP1 primers can be purchased from Bio-Rad .Purified RNAs were reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). In all, 20 ng  were isolated by density gradient using Ficoll-paque PLUS  and red blood cells were lysed by ammonium chloride potassium (ACK) lysing buffer. Cells were then immediately pelleted and stored at −80 °C until use. RNA was extracted from cells using RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and analyses by qPCR or ddPCR.All western blots and DNA gels were repeated at least three times and representative images were shown in this paper.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4