Colorectal cancer (CRC) is a prevalent malignancy worldwide and is associated with a high mortality rate. Changes in bioenergy metabolism, such as the Warburg effect, are often observed in CRC. Aldolase B (ALDOB) has been identified as a potential regulator of these changes, but its exact role in CRC cell behavior and bioenergetic homeostasis is not fully understood. To investigate this, two cohorts of CRC patients were analyzed independently. The results showed that higher ALDOB expression was linked to unfavorable prognosis, increased circulating carcinoembryonic antigen (CEA) levels, and altered bioenergetics in CRC. Further analysis using cell-based assays demonstrated that ALDOB promoted cell proliferation, chemoresistance, and increased expression of CEA in CRC cells. The activation of pyruvate dehydrogenase kinase-1 (PDK1) by ALDOB-induced lactagenesis and secretion, which in turn mediated the effects on CEA expression. Secreted lactate was found to enhance lactate dehydrogenase B (LDHB) expression in adjacent cells and to be a crucial modulator of ALDOB-mediated phenotypes. Additionally, the effect of ALDOB on CEA expression was downstream of the bioenergetic changes mediated by secreted lactate. The study also identified CEA cell adhesion molecule-6 (CEACAM6) as a downstream effector of ALDOB that controlled CRC cell proliferation and chemoresistance. Notably, CEACAM6 activation was shown to enhance protein stability through lysine lactylation, downstream of ALDOB-mediated lactagenesis. The ALDOB/PDK1/lactate/CEACAM6 axis plays an essential role in CRC cell behavior and bioenergetic homeostasis, providing new insights into the involvement of CEACAM6 in CRC and the Warburg effect. These findings may lead to the development of new treatment strategies for CRC patients. Colorectal cancer (CRC) is a prevalent malignancy with high mortality worldwide, including Taiwan. According to a report by Global Cancer Observatory, about one million new cases were diagnosed in 2020, with 5.7 billion deaths due to CRC complications . DespiteThe development of CRC is initiated by the transformation of premalignant adenomatous polyps (APs) through the adenoma-to-carcinoma sequence, which is induced by genetic mutations, inflammatory responses, growth factor expression, and activation/inactivation of cancer-associated signaling pathways , 4. MetaBioenergy is derived from anaerobic lactic acid fermentation, aerobic glycolysis, and mitochondrial oxidative phosphorylation (OXPHOS) . Cancer Previous studies suggest that Aldolase B (ALDOB) expression is associated with changes in AP or CRC bioenergetics . ElevateT > N), and the remaining patients having lower or equivalent levels (T ≤ N), as illustrated in Fig. P = 0.048) and circulating carcinoembryonic antigen (CEA) (P = 0.048).In order to investigate the potential role of ALDOB in CRC, the levels of ALDOB were assessed in patient tissues using immunohistochemical (IHC) staining. The staining intensities in both nontumor and tumor areas on a single slide were compared. Patients were then grouped based on their level of ALDOB in the tumor area, with one group having higher levels than the nontumor area (P = 0.033) and receipt of adjuvant chemotherapy (P = 0.002) were found to be linked with overall survival (OS). Multivariate analysis indicated that both ALDOB expression and patient experience with adjuvant chemotherapy were independent predictors of OS . Similarly, ALDOB expression levels (P < 0.001), gender (P = 0.022), and adjuvant chemotherapy experience (P = 0.009) were significantly associated with disease-free survival (DFS). The multivariate analysis revealed that ALDOB expression, gender, and patient experience with adjuvant chemotherapy were independent factors for DFS .Cox regression analysis was performed to investigate the association of clinicopathological factors and ALDOB levels with the prognosis of CRC patients. As presented in Table T ≤ N) were found to have a more favorable prognosis    had an unfavorable prognosis  Fig. . Using tP = 0.011). Corresponding to the protein level findings, patients with high ALDOB mRNA levels in tumor tissue (T > N) had an unfavorable prognosis  showed significantly increased ECAR (P = 0.034) and a borderline increase in OCR (P = 0.053)  and a weaker positive correlation with OCR ratios  , was observed, while the aerobic glycolytic capacity, represented as extracellular acidification rate (ECAR), was only slightly altered . A subse53) Fig. . Further41) Fig. .P > N) was associated with lower ECAR and OCR   Fig. .Previously, it was demonstrated that higher levels of ALDOB expression can boost cell proliferation . Our finGiven that higher ALDOB expression was associated with increased ECAR and OCR (with borderline significance) in tissues from CRC patients Fig. , we explAs we observed a correlation between higher tumor ALDOB expression and elevated circulating CEA levels in patients Table , we sougThe alteration of CEACAM6 and pan-CEA levels in CRC cells could be a result of ALDOB-induced disruption of bioenergetic homeostasis. To test this hypothesis, we employed inhibitors of either the OXPHOS or glycolytic pathway to induce bioenergetic alterations, and examined their impact on CEACAM6 and pan-CEA levels. Our results, depicted in Fig. To determine if the relationship between ALDOB expression and CEACAM6 or pan-CEA levels is also evident in tissues obtained from CRC patients, we conducted western blotting experiments. Our results, depicted in Fig. Given ALDOB’s involvement in the glycolytic pathway, we sought to investigate whether its overexpression would impact the metabolic reprogramming of CRC cells. To do this, we used NMR to analyze intracellular metabolite levels. Our results, depicted in Fig. To determine whether cell proliferation induced by ALDOB occurs through the secretion of specific factors such as lactate, we conducted two experiments. First, we investigated whether ALDOB-overexpressing cells could secrete agents to promote proliferation and insensitivity to anticancer drugs in parental cells. As shown in Fig. The overexpression of ALDOB has been shown to increase lactate production and secretion , which has been recognized as a hallmark of metabolic reprogramming. This modification plays a pivotal role in balancing gene expression and metabolic reprogramming in cancers, potentially through influencing protein stability , 24. In Intriguingly, to ascertain whether the increased protein stability could be linked to the state of Kla within CEACAM6, we conducted an immunoprecipitation of CEACAM6 followed by the detection of Kla using a specific antibody. Our findings, illustrated in Fig. The ALDOB protein level has been suggested as a biomarker for predicting postoperative prognosis in CRC patients, as well as the risk of colon cancer liver metastases and the response of rectal cancer patients to neoadjuvant chemoradiation therapy –21. AddiThe oncogenic properties of glycolytic pathway enzymes, such as their association with cell growth, migration, and chemosensitivity, have been studied extensively in various types of cancer . SimilarLactagenesis in lactagenic cancer cells is believed to be the primary cause and purpose of the Warburg effect . There iTo summarize, this study has demonstrated that overexpression of ALDOB in CRC cells promotes lactagenesis by regulating PDK1 activation. The secreted lactate is then transported to neighboring cells and converted to pyruvate by lactate-induced LDHB, enhancing the ability of OXPHOS in terms of basal respiration and acting as a repressor of CEACAM6 expression. Consequently, ALDOB/lactate-mediated expression of CEACAM6 promotes cell proliferation and 5-FU chemoresistance in CRC cells Fig. . These fThis study retrospectively enrolled two cohorts: cohort-1, which consisted of 428 CRC patients who underwent surgical resection, and cohort-2, which included 64 patients with AP. Of the patients in cohort-1, 299 were available for paraffin-embedded tissue sections, while the remaining 129 were available for frozen tissue. In cohort-2, all 64 patients were available for frozen tissue analysis. The IHC staining was performed on paraffin-embedded tissues, while RNA isolation, Western blot, and seahorse analysis were conducted on frozen tissues. All patient-derived tissues were retrieved from the Tissue Bank of Linkou Chang Gung Memorial Hospital, and their clinical data were reviewed, including gender, age, height, weight, body mass index (BMI), tumor location, tumor differentiation status, local invasion score, maximum tumor size, circulating CEA level, history of adjuvant chemotherapy, tumor-to-serosa distance, date of CRC diagnosed, date of CRC-related death, date of last follow-up, and date of recurrence or metastasis. This study was conducted with the approval of the Chang Memorial Hospital Institutional Review Board .Western blots were conducted following previously described methods . The rabIHC was conducted following previously described methods . A rabbiTotal RNA isolation was conducted following previously described methods . The Pri2 incubator for 30 min, and then used the Seahorse XF24 analyzer to measure OCR and ECAR simultaneously. During the measurement, the tissue was not pushed by the sensor, and we used at least three replicates from each tissue for the assays. We transferred the specimen from the colonoscopy suite or operating theater to the Seahorse platform within two hours. To detect bioenergetic alterations in cells, we seeded cells into 24 wells of an XF24-cell culture microplate at a density of 7 × 104 or 5 × 104 cells/well and incubated them in a 5% CO2/air atmosphere at 37 °C for 24 h. We then measured OCR and ECAR under basal conditions using the Mito Stress test Kit  to reveal key parameters of the metabolic function of cells. OCR was measured under basal conditions and in the presence of the ATP synthase inhibitor oligomycin (1 μM), the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy)-phenyl-hydrazone , and the respiratory chain inhibitor antimycin A (0.5 μM). We used the Glycolysis Stress test Kit  to measure ECAR under various conditions. We made three baseline recordings, followed by sequential injection of glucose (10 mM), the ATP synthase inhibitor oligomycin (1 μM), and the glycolysis inhibitor 2-deoxy-d-glucose . We performed the assays in at least three replicates and presented OCR and ECAR as absolute values, normalized by the tissue protein concentration  or cultured cell concentration .Simultaneous measurements of OCR and ECAR of patient tissues or cultured cells were performed using a Seahorse XF24 analyzer  as formerly described . In brieHT29 (CVCL-0320) and WiDr (CVCL-2760) cell lines were used in this study and maintained in RPMI-1640 and DMEM media, respectively, at standard culture conditions of 5% CO2 in a humidified 37°C incubator. Both cell lines were routinely screened to ensure they were free of mycoplasma contamination. To express ALDOB with an N-terminal myc tag, a plasmid was purchased from SinoBiological . For transient expression of ALDOB, cells were seeded 16 h prior to transfection, and 5 μg of plasmid DNA was used for transfection in 6 cm plates. MaestroFectin  transfection reagent was used according to the manufacturer’s instructions.To block OXPHOS activity, oligomycin  and Antimycin A  were used at a final concentration of 1 μM and 2 μg/mL, respectively. The glycolytic pathway was blocked using 2-DG  at a final concentration of 2 or 4 mM. To mimic extracellular secreted lactate, lactate  was used. All cells were collected for subsequent analysis 48 h after treatment.3 cells were seeded into each well of a 96-well plate and incubated for 24 h before adding Alamar Blue cell viability reagent  directly to the medium. Fluorescent metabolite quantification assays were performed daily for up to 4 days. To assess cell viability in response to anticancer drugs, 1 × 104 cells were seeded per well of a 96-well plate at least 16 h prior to treatment with 5-FU or Oxaliplatin. Following a 24 h incubation at 37 °C, the medium in each well was refreshed, and Alamar Blue cell viability reagent was added. Fluorescent metabolite quantification was performed after a further 3 h of incubation at 37 °C. The growth-promoting and anticancer drug insensitivity effects of ALDOB were also assessed using a transwell-based co-culture system and culture of parental cells in medium containing ALDOB-overexpressing cells. For the transwell-based co-culture system, 2 × 104 parental cells were seeded at the bottom of each well of a 24-well plate, and 1 × 104 cells were seeded in the transwell chamber with or without ALDOB overexpression. After 48 h of co-cultivation, the cells in the bottom were suspended with trypsin, and the total number of cells was counted. Similarly, 2 × 104 parental cells were seeded in each well of a 24-well plate and the next day, replaced with fresh medium containing medium derived from control or ALDOB-overexpressing cells previously used. After 48 h of co-cultivation, the cells were suspended with trypsin, and the total number of cells was counted.Cell proliferation rate was evaluated according to previously described methods . Specifi6 cells, either with or without ALDOB overexpression, was conducted. Tumor length and wide were measured twice weekly until sacrifice. The formulation: 3, administered twice weekly. After six 5-FU injections, mice were sacrificed, tumors were removed, weighed, and fixed in 10% formaldehyde. Paraffin embedding followed for subsequent IHC analysis. Animal protocols adhered to regulations set by the Animal Ethics Committee of Linkou Chang Gung Memorial Hospital.In this study, 36 male nude mice (6 weeks old) purchased from National Animal Center (Taiwan) were used. Of these, 20 were dedicated to the xenograft tumor growth study, with 10 for the WiDr cell group and 10 for the HT29 cell group. Subcutaneous injection of approximately 5 × 106 cells from a 10 cm culture dish were harvested and subjected to metabolite extraction in the aqueous phase. The cells were lysed with pre-chilled methanol and then mixed with chloroform and sterile water at a 1:1:1 ratio. After centrifugation, the aqueous phase was collected and concentrated using a vacuum concentrator with a lyophilizer. The lyophilized metabolites were then redissolved in NMR buffer containing a chemical shift reference. The samples were placed in a 4-inch SampleJet NMR tube and analyzed using a Bruker Avance III HD spectrometer with a 14.1-T magnet for 1H 600 MHz. Independent biological triplicates were performed for reproducibility. The acquisition and processing of data were performed using Topspin 3.6, and the experiments were automated using the IconNMR program with a standard pulse sequence (cpmgpr1d). Continuous wave irradiation was used with 25 Hz RF during the relaxation delay and mixing time, with a magnetic field Z-gradient applied for 1 min. The relaxation delay was set to 4 s, the mixing time was 10 min, and the acquisition time was 2.7 s with 128 transients of 64,000 data points recorded. The receiver gain was set to 64, and a cooling rack was used to maintain the sample at 283 K.The intracellular metabolites were analyzed using nuclear magnetic resonance (NMR), following a previously described protocol . To prepThe protocol for IFA was performed with minor adjustments as previously described . In brieLentiviral-mediated transduction of shRNA against CEACAM6 was carried out using a previously described protocol with slight modifications . In briet-test. Dichotomized data were presented as numbers and percentages (%) and compared using the chi-square test. Univariate and subsequent multivariate Cox proportional hazard models were used to assess the prognosis for clinical factors and ALDOB expression. Significant factors identified in the univariate analysis were included in the subsequent multivariate Cox proportional hazards analysis. Survival probabilities between different groups were estimated using the Kaplan–Meier method, and the log-rank test was applied. All tests were two-tailed, and statistical significance was set at P < 0.05. The Statistical Package for the Social Sciences (SPSS) statistics Version 20 was used to perform all statistical analyses.The parametric data were reported as mean ± standard deviation and compared using a two-sample ChecklistOriginal Data FileSupplemental material


Cellular senescence is an important factor in aging and many age-related diseases, but understanding its role in health is challenging due to the lack of exclusive or universal markers. Using neural networks, we predict senescence from the nuclear morphology of human fibroblasts with up to 95% accuracy, and investigate murine astrocytes, murine neurons, and fibroblasts with premature aging in culture. After generalizing our approach, the predictor recognizes higher rates of senescence in p21-positive and ethynyl-2’-deoxyuridine (EdU)-negative nuclei in tissues and shows an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies. Evaluating medical records reveals that higher rates of senescent cells correspond to decreased rates of malignant neoplasms and increased rates of osteoporosis, osteoarthritis, hypertension and cerebral infarction. In sum, we show that morphological alterations of the nucleus can serve as a deep learning predictor of senescence that is applicable across tissues and species and is associated with health outcomes in humans. Senescent cells are typically identified by a combination of senescence-associated markers, and the phenotype is heterogeneous. Here, using deep neural networks, Heckenbach et al. show that nuclear morphology can be used to predict cellular senescence in images of tissues and cell cultures. Due to its critical role in disease etiology, senescence is increasingly recognized as a target for pharmaceutical intervention4. Senescence also serves as a biomarker for aging5, possibly providing a more nuanced measure of age-related health beyond chronological age. However, the role of senescence in human health is not clearly understood. Senescent cells present a complex and diverse phenotype, which varies substantially by cell type and source7. There is considerable overlap between molecular factors that associate with senescence, DNA damage, inflammation and other processes9, and no single marker reliably and consistently identifies senescence12. Importantly, senescent cells often exhibit an altered morphology, including expanded nuclei14, making senescence amenable to analysis with computer vision and machine learning methods15.Cellular senescence is widely recognized as a fundamental process in aging, both as a primary causal factor in the decline of tissue homeostasis and as a consequence of other aging processes such as inflammation and DNA damageInk4a, p21Cip1, p53 and DNA damage markers γH2AX and 53BP1 foci counts. Our senescence predictor was developed using normal human fibroblasts but also identifies increased senescence for multiple types of premature aging diseases, including Hutchinson–Gilford progeria syndrome, ataxia telangiectasia and Cockayne syndrome. We also evaluated mouse astrocytes and neurons and found increased senescence in cells subjected to ionizing radiation (IR), confirming its relevance to different cell types and organisms. These methods were further applied to H&E-stained mouse liver and human dermal tissues, predicting an increase in senescence with age. Using the Danish National Patient Register, which records all ambulatory and inpatient contacts with Danish hospitals, we investigated how predicted senescence relates to human disease. In our study of 169 individuals, we found a significant inverse relationship between malignant neoplasm incidence and predicted senescent cells, which fits the hypothesis that senescence is a mechanism to limit cancer18. Although oncogenic events are associated with the formation of senescent cells17, we speculate that individuals with higher propensity toward developing senescent cells have reduced formation of malignant neoplasm and are at lower risk of cancer. We also found weaker associations between predicted senescence and other conditions, including osteoporosis, osteoarthritis, hypertension, cerebral infarction, hyperlipidemia, hypercholesteremia, hearing loss, dyspnea and sciatica.We present deep learning models that can predict cellular senescence with high accuracy based on nuclear morphology. Notably, predicted senescence correlates substantially with senescence-associated β-galactosidase , p16Ink4a, p21Cip1, p53 and interleukin-6 (IL-6) by immunohistochemistry and/or qPCR and found that IR led to a significant increase in these markers  . We found both control and RS groups had no correlation, but IR had a negative correlation  can be used to filter samples and reduce ambiguous predictions by requiring higher mean probability. Using FlipOut nodes29 in Tensorflow Probability, we converted Xception and InceptionV3 and trained using our senescent nuclei data set. Our BNN of Xception produced accuracy of 86% on raw images . Our base data set including control, RS and IR lines were examined for damage foci using high-content microscopy, where we found the mean count for controls to be below 1 for each marker, whereas RS had 4.0 γH2AX and 2.0 53BP1 foci and IR had 3.4 γH2AX and 3.0 53BP1 foci  and 34.2% for testis (P = 2.6 × 10−8)  Fig. . We also−8) Fig. . To deteied Fig. .Fig. 5Se39. With these thresholds, the percentage of senescent cells per mouse increased with age , and evaluated each of them. PCNA staining confirmed that each drug treatment led to a senescent state , whereas other models showed no difference  showed no separation between p21Cip1 positivity and p21Cip1 negativity, indicating that they are picking up on other type-specific aspects of senescence.To determine if the predictor could be used with human dermis, we analyzed samples from an independent data setnce Fig. . As the 43, so we selected thresholds to calibrate the model with 0.7 for RS and 0.85 for IR, leading to an overall predicted percentage of ~6% and showing an age-dependent increase in percent senescence , indicating that each group has a similar proportion of cell types.To further investigate if the predictor could be applied in a clinical context, we tested the algorithm on human skin samples of 169 individuals aged 20–86 years. The senescence classifier was used to evaluate the dermal nuclei from biopsy samples, stained with H&E and imaged in a slide scanner at ×20. We used U-Net to detect nuclei, extracted nuclear regions and fully normalized images of the nuclei Fig. . We firsnce Fig. . Both IRnce Fig. . We alsonce Fig. . Interesnce Fig. . CompariP values of 0.002 and 0.005, respectively. Narrowing down the analysis, we determined the association was based on malignant (versus benign or unknown) codes within ICD-10 Chapter II Neoplasm with IR P value at 0.018 and RS at 0.058. We also scanned individual ICD-10 clinical codes and found several other conditions associated with senescence, including osteoporosis, osteoarthritis, hypertension, cerebral infarction, hyperlipidemia, hypercholesteremia, sciatica, dyspnea and hearing loss, which were all significant when evaluated individually but nonsignificant when applying multiple test correction, such as the Bonferroni method  diagnosis codes collected in the Danish National Patient Register from 1977 to 2018 for all the individuals in the study. We looked for associations between individuals with diagnosed conditions grouped by ICD-10 chapters and predicted senescence above or below the age-dependent mean . We used the chi-squared test and Fisher’s exact test for the frequency of occurrence between the two groups Fig. . Remarkahod Fig. . All of In this paper, we present a neural network classifier that can predict cellular senescence based on nuclear morphology. Trained on fibroblasts maintained in cell culture, the classifier achieves very accurate results, which was confirmed by applying it to independent cell lines. By training additional models on samples with reduced features, we infer that the shape of the nucleus alone provides a signal to indicate senescent state. DAPI-stained nuclei with background removed, size standardized and internal structure masked are still classified with high accuracy. These feature reduction methods serve a secondary purpose, making a model robust to technical variation; our neural network trained on reduced samples can make predictions on nuclei that were prepared in other experimental and imaging contexts. Indeed, the predictor distinguished senescent astrocytes and neurons, predicted an age-related increase in senescent liver cells and confirmed senescence in cell lines from patients suffering from premature aging. Although it is still debated if universal markers of senescence exist, our findings suggest that at least morphological alterations in nuclei may be common across some tissues and species.We present several predictor models, including those that combine IR, RS and other methods, and those that specialize on each for improved accuracy. The base model trained on IR and RS can identify either type along with senescence induced by doxorubicin, indicating that the predictor has identified features found in multiple types related to DNA damage. Our base model did not accurately identify ATV/r and antimycin A, but a new model trained on all five methods could identify senescence induced by these diverse mechanisms. The unified model could be identifying a common signature or simply recognizing multiple phenotypes.We evaluated the predictor with two tissues, mouse liver and human dermis, which include heterogenous cell types. It is possible that age-associated changes in cell types, such as an increase in tissue-resident macrophages, could influence the predictor. For the liver, this is likely not the case, as segmentation was specifically trained on the distinct nuclear morphology of hepatocytes. Nevertheless, age-associated changes in tissue composition should be considered when interpreting the outcome of the algorithm in vivo. In general, it is challenging to assess senescence in tissues owing to the lack of universal markers, the expression of senescence-associated markers by quiescent cells and some nonsenescent cell types and the diverse variations of senescence and the SASP. Although our deep learning models predict higher rates of senescence in p21-positive and EdU-negative nuclei and show an increased rate with age, their accuracy in tissues is unclear. Indeed, the true rate of senescence in tissues is poorly characterized and can perhaps only be approximated by combining multiple traditional markers. Accordingly, when evaluating tissues, we expect lower accuracy but the utility of the method can be increased by focusing on restricted cell types or evaluating cell-type proportions. Further, large image data, such as full slide images as well as a considerable number of samples, may be needed to address heterogeneity.18. Further, premalignant tumors express markers of senescence, which are absent in malignancies, and malignant tumors can regress and undergo senescence by switching off oncogenes17, supporting the protective role of senescence in blocking the progression of neoplasms to malignancies. In addition, loss of central senescence inducers such as p16Ink4a is very common in many cancer types44. Of note, there is also evidence suggesting that cellular senescence promotes malignancy through the inflammatory senescence-associated secretory phenotype or SASP45, that senescent cells may appear in areas where tumors tend to subsequently develop46, and that senescent cells and SASP induced by cancer treatment led to worse survival and healthspan47. Although the role of senescence in cancer is highly complex, our results based on clinical data support the overall protective role for senescence in human health with regard to cancer. We also found several other conditions associated with senescence48, including osteoporosis49, osteoarthritis50, hypertension51, cerebral infarction52, hyperlipidemia, hypercholesteremia, dyspnea and sciatica.Given these caveats, our data show that individuals with a predicted higher rate of senescent cells have reduced neoplasms and malignant cancer. This finding is consistent with the notion that senescence is a mechanism to control cancer development by limiting uncontrolled proliferation54, and we confirmed this in our cell culture data set. On a per-nuclei basis, we found a moderate correlation between area and predicted senescence. However, due to our size standardization, it is unlikely this classic feature is the primary signal for our deep learning model. We also identified convexity and aspect ratio as indicators of senescence, finding moderate correlation with predicted senescence. Interestingly, we found no increase in area with age in the human dermis, but we found a significant increase in aspect ratio and a significant decrease in convexity, indicating that nuclei become stretched and irregular with advancing age in humans. These observations confirm that size standardization is necessary to generalize our neural network classifier and also demonstrate the value of our feature-neutral approach, where the neural network is trained to identify senescence from rich image data that is later reduced through feature removal.We also investigated how our deep learning predictor results correspond to other measures of senescence. Nuclear area is known to expand during senescenceIn sum, our deep neural network model is capable of accurately predicting the senescent state from nuclear morphology using several imaging techniques and has been demonstrated with diverse applications. We applied the predictor to human skin samples and observed an age-dependent increase in senescence. Remarkably, individuals who appear to have higher rates of senescent cells show a reduced incidence of malignant neoplasms, supporting the hypothesis that senescence is a mechanism to limit cancer. Further, we find association between predicted senescent cell burden and other conditions, including osteoporosis, osteoarthritis, hypertension, cerebral infarction, hyperlipidemia, hypercholesteremia, dyspnea and sciatica. These findings support the idea of a dichotomy in aging, where senescence limits age-associated proliferation while promoting age-associated degeneration.All experiments and research conducted within this work comply with all relevant ethical regulations.2 either in 1:1 mix of DMEM GlutaMAX  and F-12 media  for AG08498, GM22159 and GM22222 or in EMEM media  for GM03349 and GM05757. Fibroblasts derived from Hutchinson–Gilford progeria syndrome patients included AG06917 , AG06297  and AG11513 . Fibroblasts sampled from ataxia telangiectasia and Cockayne syndrome patients were GM03395  and GM01428 , correspondingly. Cells were cultured at 37 °C and 5% CO2 in minimum essential medium (MEM) . Freshly isolated primary neurons and mouse astrocytes were kindly provided by the Department of Drug Design and Pharmacology, University of Copenhagen. Cells were cultured at 37 °C and 5% CO2 in DMEM GlutaMAX . All media were supplemented with 10% fetal bovine serum  and 100 U ml−1 penicillin-streptomycin .All human-derived primary skin fibroblast cells were purchased from Coriell Institute. Control fibroblasts included AG08498 , GM22159 , GM22222 , GM03349  and GM05757 . Cells were cultured at 37 °C and 5% CO2(cell number during harvesting/cell number during seeding). The experiment was terminated when the population doubling level reached zero.To achieve RS, control fibroblasts at early passages were seeded in T25 cell culture flasks  and cultured over 32 weeks. After each splitting, cell number was recorded and population doubling level was calculated as log55. Briefly, control fibroblast cells at early passages were seeded in 96-well plates  in a density of 2,000 cells per well. The day after, cells were either exposed to 10 Gy IR or treated with 250 nM doxorubicin for 24 h and cultured for the next 9 days. Medium was replaced every 2 days. Three days before radiated or doxorubicin-treated cells reached senescence state, fibroblast cells from the same stock were seeded  as mock-irradiated or DMSO-treated controls. For tracking proliferating fibroblasts, control cells were stained with CFSE using Vybrant CFDA SE tracer kit  according to the manufacturer’s protocol. The control cells were subsequently added to the senescent cells. The co-cultured proliferating and senescent cells were cultured for 24 h before fixing. Mitochondrial dysfunction-induced senescence was achieved by treating control fibroblast cells with 250 nM antimycin A every 2 days within 10 days. Then, 25 μM ATV/r was given to control fibroblast cells every 2 days within 14 days to develop senescence phenotype. Corresponding DMSO-treated controls were cultured in parallel and seeded in a 96-well plate 3 days before terminating the experiment. Temporary cell cycle arrest in quiescent fibroblasts was induced by culturing cells in 0.2% FBS-containing media for 14 and 21 days56. On day 11 for the 14-day treatment and day 18 for the 21-day treatment, media containing 10% serum was added back to half of cells to restore their proliferative capacity.Induction of cellular senescence by IR, doxorubicin, antimycin A and ATV/r was performed according to Neri et al.55: CDKN2A (p16Ink4a): forward: 5′-GAG CAG CAT GGA GCC TTC-3′, reverse: 5′-CGT AAC TAT TCG GTG CGT TG-3′; CDKN1A (p21Cip1): forward: 5′-TCA CTG TCT TGT ACC CTT GTG C-3′, reverse: GGC GTT TGG AGT GGT AGA AA; IL-6: forward: 5′-CAG GAG CCC AGC TAT GAA CT-3′, reverse: GAA GGC AGC AGG CAA CAC; GAPDH: forward: 5′-GTC AGC CGC ATC TTC TTT TG-3′, reverse: 5′-GCG CCC AAT ACG ACC AAA TC-3′.Radiated and control fibroblast cells were lysed using TRIzol reagent (Ambion by Life Technologies), and RNA was isolated using Direct-zol RNA miniprep Plus (Zymo Research). RNA was reverse transcribed into complementary DNA using High-capacity complementary DNA reverse transcription kit (Applied Biosciences) according to manufacturer’s protocols. Next, RT-qPCR was performed to detect mRNA levels of senescence markers using StepOnePlus Real-Time PCR System (Applied Biosystems). Relative gene expression was calculated based on obtained Ct values, normalized to housekeeping gene GAPDH and expressed as fold changes compared to nonirradiated control. The following specific primer sequences were usedIn total, three independent sets were performed in GM22159 cells.Ink4a, p21Cip1, p53 and PCNA, fibroblast cells were washed once with warm PBS and fixed in 4% paraformaldehyde solution for 15 min, followed by a permeabilization step with incubation for 10 min in PBS-0.1% Triton X-100. Blocking was performed in 1% BSA-PBS-0.1% Tween 20 overnight at 4 C. The next day, cells were incubated with primary antibodies  for 1 h at room temperature, washed three times with PBS-T and incubated with secondary antibodies  for 1 h at room temperature. Cells were incubated with DAPI solution  for 10 min and stored in PBS at 4 °C until the analysis.For detection of persistent DNA damage foci and fluorescence intensity levels of p162. Cells were co-stained with DAPI solution  for 10 min. Plates were stored in PBS at 4 °C until the analysis.EdU staining was detected using EdU Click 488 kit  according to the manufacturer’s protocol. For all EdU-based experiments, cells were incubated with 10 μM EdU in cultured media for 1 h at 37 °C and 5% COSA-β-gal was detected using senescence cells histochemical staining kit  according to manufacturer’s protocol. Cell colonies were imaged using INcell analyzer 2200 high-content microscopy at ×20 magnification to produce 1199 images with 2,048 × 2,048-pixel resolution. Due to system constraints for object detection, each image was split into four tiles of 1,024 × 1,024-pixel resolution.−1; Thermo Fisher Scientific) at 37 °C for 30 min. Propidium iodide  was added and incubated for 30 min at 37 °C. Cell cycle status was determined by flow cytometry .Fibroblast cell lines AG08498, GM22159 and GM22222 were seeded in six-well cell culture plates, and 24 h later, DNA-damage induced senescence was conducted as described above . After 9 days, cells were harvested by trypsinization and washed twice with PBS. Cells were fixed by adding dropwise ice-cold 70% ethanol while mixing the cells gently on a vortex mixer. Thereafter, the cells were incubated for 30 min on ice and washed twice in PBS. Fixed cells were incubated with RNase , producing output images of predicted nuclei regions. The images with predicted nuclei were scanned for recognition regions of area between 500 and 15,000 pixels. Each detected nucleus was extracted along with its surrounding context as a centered 128 × 128-pixel region and used to assemble a base library of 95,152 nuclei. In addition, the recognition region itself was cutout, providing a two-color reduction of the detected nuclei, and assembled into a secondary library of nuclei masks.A base library was prepared using control, irradiated (IR) and cells serially passaged until they reached senescence (RS). A deep neural network model was applied to detect DAPI-stained nuclei. The samples were used to build a training set for nuclei recognition. Several images were selected arbitrarily from each group for a total of ~20 samples, and all nuclei in the training samples were annotated by selecting the nuclear region. U-Net, a 23-layer fully convolutional network for image segmentation, was trained using the samples, learning to associate the DAPI images with annotation masks indicating nuclear regions. Our implementation of U-Net is largely based on the original U-NetAn analysis of the nuclei was performed to assess morphological properties. The two-color mask library was used, as it provided a universal representation of the detected nuclei (with U-Net detector models that have good coverage of the nuclei region). Nuclear morphology was assessed using several metrics, including area, perimeter, moments, convexity, and aspect ratio. Convexity is the ratio of convex hull perimeter to perimeter, which provides a size-neutral measure of boundary regularity. The convex hull is a polygon that connects the outer edges of nuclei like an envelope.−4 for ten epochs, in which time accuracy rapidly converged to a steady level. In addition, a simpler custom model was tested, with three convolutional layers with ReLU activation and two dense layers with L1/L2 regularization of 0.05/0.05 and 30% dropout. This model required 713,296 parameters. For both network designs, we trained with raw images along with several modified image sets, where the background was removed, the nuclei were size normalized and the inner details of nuclei were entirely masked , whereas additional cell lines (GM22222 and AG08498) were used for testing. Training samples were randomized and split into 80% for training and 20% for validation. Due to experimental setup, the sample classes are unbalanced, with 75.2% control, 11.2% RS and 13.6% IR. The samples were balanced during training by applying class weights with inverse proportion to the class abundance . Image samples were normalized for brightness/intensity by adjusting each image’s mean intensity to 0 and standard deviation to 1. Augmentation was also applied during training, randomly modifying samples . For each epoch, one augmentation cycle was performed. Training was done with Xception, a 48-layer model, initialized with ImageNet weights but set to allow weight adjustment of all layers during training. The top layer was replaced by a layer of one-hot nodes to indicate the state as controls or senescent. With this minor adjustment, the model provided 37,640,234 trainable parameters. Training was done using Adam with the learning rate set to 1 × 10ked Fig. . All thr29, which assumes that the kernel and bias are drawn from a normal distribution. During a forward pass, kernels and biases are sampled from posterior distribution. Targets were encoded as above, and the loss function used was cross-entropy plus Kullback–Leibler divergence divided by the number of batches. We also partially converted Xception to a BNN by replacing all dense and convolutional layers to FlipOut nodes, leaving separable convolutions unconverted, as a FlipOut version was not available. In addition, we fully converted InceptionV3 for evaluation. Inference was done by evaluating the model 20 times to produce a distribution of predictions and then taking the mean probability for each sample.We used Tensorflow Probability to create a BNN. We first converted the simple custom model, replacing nodes with the comparable FlipOut versionn = 1 − 1/e. With each instance trained on a different subset of samples, this method produces multiple models that in theory can specialize to different sets of data.To improve accuracy and provide a more robust solution, we also worked with an ensemble of deep learning models. This method used ten models of Xception, each trained on the same data set with different random weight initialization. To generate predictions, each model instance was applied, and the results combined by taking the mean prediction. We also tried bagging, also known as bootstrap aggregation. Similar to the deep ensemble, this method trains different model instances with bootstrap selection of samples for The following software tools were used for the predictor and related analysis: python 3.8.10, tensorflow-gpu 2.4.1, tensorflow-probability 0.11.1, scipy 1.6.2, statsodels 0.12.0, scikit-learn 0.23.2, pandas 1.1.1, seaborn 0.11.1 and numpy 1.21.6, R 4.0.2f-tests to evaluate differences in the means, followed by pairwise tests using Tukey’s honest significant difference to calculate P values between groups. Linear regression methods were evaluated with R and P value statistics. Groups of patients were compared using the chi-squared test and Fisher test to detect significant differences between frequencies. Correlation was evaluated using the Pearson colocalization coefficient. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Data distribution was assumed to be normal, but this was not formally tested.All comparisons between groups of samples were made using one-way analysis of variance T02530 (skin on penis), T76330 (foreskin), T80200 (mons pubis), T02471(skin on nates), T02480 (skin on abdomen), T02430 (skin on breasts) and one or more procedure code: P30620 (resect), P306X0 (ectomy preparation), P30611 (excision biopsy) and one or more morphology code: M87400 (junction naevus), M87500  and M87600 (compound naevus).The individuals were sampled from patients for whom samples of naevi on non-sun-exposed skin had undergone pathology without malignant findings at a major pathology department in Copenhagen. The patient sample was selected to have flat distribution of age. We selected patient samples from the Danish National Register of Pathology requisitioned in 2007–2017 and coded with one or more PatoSNOMED topology code: We collected ICD-10 diagnosis codes from the Danish National Patient Register in the period 1977-2018 of each of the patients in this study. We further grouped diagnoses into each of 21 ICD-10 chapters. We calculated the linear regression residuals of the relationship between age at pathology examination and the predicted senescent cell load  for each of the patients. We then constructed contingency tables counting the number of patients with and without a specific diagnosis and with a predicted senescent cell load above or below the age-dependent average. We used Pearson’s chi-squared test and Fisher’s exact test to determine whether patients with a predicted senescent cell load above or below the age-dependent average were associated with a higher or lower incidence of specific diagnosis codes (or diagnosis within a specific ICD-10 chapter.)For liver histology studies, 15 male C57BL/6J mice were acquired from Janvier Labs. Animals arrived at 5–8 weeks of age and were housed in a controlled environment . Stratification and randomization into individual diet groups were based on baseline body weight. Mice had ad libitum access to tap water and chow . Animals were aged to 48, 58 and 78 weeks, with five animals per group. For EdU proliferation in mouse tissues, six male and six female 12-month-old C57BL/6NRj mice (Janvier Labs) were bred in-house at the Department of Experimental Medicine, University of Copenhagen and maintained in a climate-controlled room with a 12-h light/dark cycle and ad libitum access to standard laboratory chow  and water.All animal experiments were approved by the Institutional Animal Care and Use Committee at MedImmune and/or the Danish Animal Experiments Inspectorate (licenses 2017-15-0201-01378 and 2017-15-0201-01321) and performed in accordance with internationally accepted principles for the use of laboratory animals including the European directive 2010/63/EU on the protection of animals used for scientific purposes. Work on human samples and clinical records were approved by the National Committee on Health Research Ethics (H-19078472) and the Danish Data Protection Agency (514‐0226/18‐3000).Cip1 . Slides were scanned by ScanScope AT System (Aperio).Terminal liver samples were dissected from the left lateral lobe immediately after killing the animal and subsequently fixed overnight in 4% paraformaldehyde. The liver tissue was then paraffin-embedded and sectioned at a thickness of 3 µm. Sections were stained with H&E (Dako), or DAPI (Invitrogen) co-stained with p21−1 EdU (Baseclick) dissolved in sterile PBS, and 4 h later, mice were euthanized and tissues collected. Testis and epididymis were dissected free from the epididymal fat pad, whereas dorsal skin was shaved, visible fat was removed and samples were fixed in neutral buffered formalin for 48 h. Sections were paraffin-embedded and sectioned by the Core Facility for Integrated Microscopy at the University of Copenhagen. Tissue sections were deparaffinized in xylene and rehydrated before fixation in 4% cold paraformaldehyde for 15 min, washed with PBS with 1% BSA and permeabilized in 0.5% Triton X-100 for 20 min. EdU staining was carried out according to manufacturer’s protocol , co-stained with 1 μg ml−1 DAPI and coverslips mounted with Prolong Gold Anti-Fade (Thermo Fisher Scientific). Whole slides were imaged on a Zeiss Axioscan.Z1 (Zeiss Microscopy), equipped with a Plan Apo 20×0.8 NA objective and a Semrock F66-887 multiband filter cube or a NanoZoomer-XR (Hamamatsu).EdU incorporation in mice was assessed by intraperitoneal injection of 50 mg kgFurther information on research design is available in the Supplementary InformationSupplementary Table 1Reporting Summary


Transcatheter annuloplasty is meant to target annular dilatation and is therefore mainly applied in functional tricuspid regurgitation (TR). Due to recent recognition of varying disease pathophysiology and differentiation of ventricular and atrial functional TR (VFTR and AFTR), comparative data regarding procedural success for both disease entities are required.n = 35, 53.8%) and AFTR . Procedural success was assessed by comparing changes in annulus dilatation, vena contracta (VC) width, effective regurgitation orifice area (EROA), as well as reduction in TR severity.In this consecutively enrolled observational cohort study, 65 patients undergoing transcatheter annuloplasty with a Cardioband® device were divided into VFTR  vs. 12 mm (7–14), p = 0.868], and EROA reduction  were reported. Improvement by at least two grades  and three grades  was similar in VFTR and AFTR, respectively. No significant difference in the accomplishment of TR grade of ≤2  was noted.Overall, improvement of TR by at least two grades was achieved in 59 patients (90.8%), and improvement of TR by at least three grades was realised in 32 patients (49.2%). Residual TR of ≤2 was observed in 52 patients (80.0%). No significant differences in annulus diameter reduction [VFTR: 11 mm (9–13) vs. AFTR: 12 mm (9–16), According to our results from a real-world scenario, transcatheter annuloplasty with the Cardioband® device may be applied in both VFTR and AFTR with evidence of significant procedural TR reduction. Tricuspid regurgitation (TR) is on the verge to become one of the major valvular heart diseases in high-income countries equally affecting morbidity and mortality , 2. EpidAlthough unappreciated for a long time due to poor surgical outcomes with high periprocedural mortality, recent advancements in interventional therapy have shed new light on different pathophysiologies driving disease progression as they also seem to further impact mortality , 7. MajoThis observational multicentre study was conducted at two tertiary care facilities of the Charité—Universitätsmedizin Berlin (Campus Benjamin Franklin and Campus Mitte) in Germany. All patients, who received catheter-based annuloplasty for symptomatic TR, have been screened and consecutively enrolled since the beginning of the dedicated institutional TR program in 2019. Overall, 67 patients were treated based on interdisciplinary heart team decision between February 2019 and January 2023. One patient was excluded from analysis due to a predominantly primary TR with an anterior leaflet prolapse, while another patient was excluded due to a mixed aetiology of TR. Patients with simultaneous finding of severe mitral regurgitation and TR received treatment for mitral regurgitation first and then transcatheter annuloplasty for TR at a later stage (>2 months later). The Institutional Review Board/ethics committee approved the conduction of this trial at each campus (EA4/013/21 and EA1/005/23).Transthoracic and transoesophageal echocardiographies were conducted in every patient as part of routine clinical examination to evaluate the necessity and feasibility of tricuspid valve intervention. Left ventricular ejection fraction (LVEF), tricuspid annular plane systolic excursion (TAPSE), fractional area change (FAC), tenting height, and the calculated systolic pulmonary artery pressure were all assessed during transthoracic echocardiography . TentingAnnulus measurements and assessment of TR severity [including vena contracta (VC) width and effective regurgitation orifice area (EROA)] were performed at the beginning and end of each procedure by transoesophageal echocardiography in the presence of general anaesthesia. Grading of TR severity was conducted according to recently proposed algorithms ranging from “none/trace” to “torrential” . To respAetiology of TR was classified either as primary, cardiac implantable electronic device (CIED)-related, atrial functional, ventricular functional, or mixed. Primary TR was defined by the presence of organic abnormalities to valve morphology, such as prolapse or flail leaflet, resulting in malcoaptation or leaflet deficiency. TR was declared as CIED-related, whenever leaflet impingement or perforation by pacemaker or defibrillator leads was observed. Differentiation of functional TR was achieved by an integrative assessment of multiple parameters. TR was classified as AFTR, whenever right atrial enlargement with consequent tricuspid annulus dilatation was suspected as the main driver for development of TR. These patients typically yielded preserved right ventricular (RV) function, absence of severe RV remodelling (triangular-shaped), and no or only mildly elevated pulmonary artery pressures . On the t-tests or Mann–Whitney U tests, according to their distribution. Differences between categorical variables were assessed by the Chi square or Fisher's exact test, whenever applicable. Univariate calculations for good response to therapy (improvement by at least three grades) were conducted by using the same tests. Figures were designed with GraphPad PRISM version 5 , and statistical analyses were conducted by using IBM SPSS version 21 . p-Values of 0.05 or less were considered significant.Continuous variables were described as median and interquartile range and categorical variables as number and percentage. Distribution of continuous variables was assessed by the Shapiro–Wilk test and inspection of quantile–quantile plots. Calculation of differences in continuous variables was either conducted by n = 46, 63.1%) were female with a median age of 80 (interquartile range: 77–83) years. All enrolled patients were found to be symptomatic with dyspnoea at exertion being the most relevant clinical symptom . The median LVEF was 53% (46–61), while TAPSE and FAC, as markers for right heart function, were 19 mm (17–22) and 37.5% (33.7–41.7), respectively. Overall, the tenting height was 6 mm (3–11), and the calculated systolic pulmonary pressure was 45 mmHg (38–56).All baseline characteristics are displayed in p = 0.017; FAC: 33.7% (28.0–38.3) vs. 39.3% (36.7–43.6), p < 0.001], respectively. Furthermore, RV area measurements were significantly enlarged in patients with VFTR . The midventricular RV diameter was more pronounced in VFTR , while the tenting height was significantly lower in AFTR . No significant differences regarding the symptomatic state of the patients as well as medical pre-treatment were observed.Differences in baseline criteria between VFTR and AFTR were mainly restricted to echocardiographic parameters. RV function was significantly better in AFTR  and EROA [0.74 cm2 (0.60–1.30)]. Cut-to-suture time was 169 min (144–230), and fluoroscopy time was 3,770 s . Implantation of the device was successful in all but one patient , in whom implantation was not attempted after right coronary artery dissection at the beginning of the procedure requiring cardiopulmonary resuscitation. Persistent injury to the right coronary artery (either stenosis or perforation) necessitating percutaneous intervention occurred in three (4.6%) patients and transient bradycardia in four (6.2%) patients. One (1.5%) major access site complication led to postponement of Cardioband® implantation. Improvement by at least two grades was achieved in 59 (90.8%) patients, and improvement by at least three grades was realised in 32 (49.2%) patients. TR of grade 2 or less was accomplished in 52 (80%) of patients (Procedure-associated data and their differences between VFTR and AFTR are displayed in patients . No casep = 0.050] and significantly reduced EROA . Nonetheless, there were no significant differences in annulus diameter reduction , VC reduction , and EROA reduction . Furthermore, no differences regarding cut-to-suture time (p = 0.200), fluoroscopy time (p = 0.095), and radiation exposure (p = 0.257) were reported. Improvement by at least two grades  and three grades  were similar in VFTR and AFTR, respectively. No significant difference in the accomplishment of TR grade of 2 or less [21 (70.0%) vs. 31 (88.6%), p = 0.118; Patients classified as AFTR were found to have numerically higher dilated annuli [42 mm (39–44) vs. 43 mm (40–49), p = 0.612), LVEF (p = 0.675), TAPSE (p = 0.440), right atrial volume (p = 0.277), diastolic RV area (p = 0.516), systolic RV area (p = 0.624), FAC (p = 0.593), calculated systolic pulmonary artery pressure (p = 0.572), tenting height (p = 0.756), CIED leads (p = 0.302), nor annulus diameter (p = 0.702) were found to predict a good procedural outcome.To assess predictors for good response to therapy, multiple parameters were investigated regarding their predictive value for TR reduction by at least three grades. Unfortunately, neither female sex (Over the past few years, several catheter-based technologies targeting TR have become available . Yet, thCurrently, there is only a single publication, which has investigated procedural results of tricuspid valve intervention distinguished by aetiology . SimilarOur findings regarding the procedural success in treatment with transcatheter annuloplasty are in line with this study. Even though patients suffering from VFTR were found to have more pronounced forms of TR at baseline, as also expressed by baseline EROA, no significant difference was observed in the changes of VC width and EROA due to transcatheter annuloplasty. The initial difference in severity might also explain the tendency of more frequently achieving moderate or less TR in AFTR. Yet, no discrepancy in reduction by at least two or three grades between both aetiologies was determined. Baseline characteristics significantly differed in terms of RV function, volumes, pulmonary artery pressure, and leaflet tenting height in our cohort, which are by definition ought to be more impaired in VFTR. Furthermore, none of these parameters independently predicted the procedural results, supporting equal efficiency of transcatheter annuloplasty in both cohorts.In contrast to ventricular dilatation causing annular enlargement and leaflet tethering in the VFTR cohort, the term AFTR suggests a dilatation of the right atrium as a major component in the development of TR. However, right atrial volumes were comparable in both groups, supporting the idea of an existing continuum between both entities. Since impairment of RV function alone will almost certainly increase atrial volumes, an atrial component of TR will be present in most of VFTR patients.From a pathophysiological point of view, and as demonstrated by our data, direct annuloplasty is able to reverse annular dilation, but not leaflet tethering as the second component of TR. According to these considerations, we expected more pronounced TR reduction with the Cardioband® device in the AFTR cohort. In fact, in terms of postprocedural VC, AFTR actually showed better results compared with their ventricular functional counterparts. However, as already mentioned earlier, this result has to be interpreted in the context of a trend towards increased VC values at baseline in the VFTR group. As stated above, the total VC reduction after the procedure was comparable in both groups. It is also important to mention that mid- and long-term postprocedural remodelling may differently behave in VFTR and AFTR. Therefore, investigation of follow-up data may be crucial to better clarify the effect of annuloplasty in both cohorts.Overall, a reduction in TR severity of at least two degrees was achieved in more than 90% of the cohort. Although 80% of the cohort presented with advanced stages of the disease displaying massive or torrential TR, a TR reduction to moderate TR or less after the procedure was still accomplished in 80% of the patients. These results are comparable to recent investigations, which first described the procedural success of interventional annuloplasty . DespiteDespite lack of follow-up data, this study represents a hypothesis-generating investigation in the largest so far reported patient cohort addressing the procedural outcome after Cardioband® in AFTR and VFTR patients. It remains a major difficulty to differentiate later stages of atrial functional TR, which also include RV remodelling due to permanent volume overload of the right heart. Regarding our study, these patients were classified as VFTR. Furthermore, the presence of significant TR may lead to an underestimation of calculated pulmonary artery pressure. Other limitations include the relative small sample size, the absence of mid-term outcome as well as any functional or mortality data, and a lack of a standardised algorithm to classify both forms of functional TR.In conclusion, this is the first analysis showing that direct tricuspid annuloplasty might be able to reverse annular dilatation in AFTR and VFTR to a similar extent with a significant decrease in TR severity. We therefore conclude that transcatheter annuloplasty with the Cardioband® device may be applied in both groups of patients with significant evidence of procedural TR reduction according to our results from a real-world scenario. However, further studies including follow-up investigations are necessary to confirm our results.


Prolonged mechanical ventilation is associated with significant morbidity in critically ill pediatric patients. In addition, extubation failure and deteriorating respiratory status after extubation contribute to increased morbidity. Well-prepared weaning procedures and accurate identification of at-risk patients using multimodal ventilator parameters are warranted to improve patient outcomes. This study aimed to identify and assess the diagnostic accuracy of single parameters and to develop a model that can help predict extubation outcomes.This prospective observational study was conducted at a university hospital between January 2021 and April 2022. Patients aged 1 month to 15 years who were intubated for more than 12 h and deemed clinically ready for extubation were enrolled. A weaning process with a spontaneous breathing trial (SBT), with or without minimal setting, was employed. The ventilator and patient parameters during the weaning period at 0, 30, and 120 min and right before extubation were recorded and analyzed.P = 0.03], >3 ventilator days , occlusion pressure (P0.1) at 30 min ≥0.9 cmH2O , and exhaled tidal volume per kg at 120 min ≤8 ml/kg ; all of these predictors had an area under the curve (AUC) of 0.72. A predictive scoring system to determine the probability of respiratory support escalation was developed using a nomogram.A total of 188 eligible patients were extubated during the study. Of them, 45 (23.9%) patients required respiratory support escalation within 48 h. Of 45, 13 (6.9%) were reintubated. The predictors of respiratory support escalation consisted of a nonminimal-setting SBT [odds ratio (OR) 2.2 , The proposed predictive model, which integrated both patient and ventilator parameters, showed a modest performance level (AUC 0.72); however, it could facilitate the process of patient care. However, prolonged intubation can lead to several complications such as nosocomial infections, airway trauma, and increased healthcare utilization . The treTo date, there is no definite standard weaning method or extubation guidance for the pediatric population. An earlier study assessed the effects of the parameters of both pre-extubation patients and ventilators; however, it did not demonstrate any cutoff value or useful clinical implications for bedside care . SeveralThe factors effecting extubation outcome may include many aspects namely respiratory muscle function, pulmonary function, sedation level, risk of airway edema, and systemic condition. Previously, predictors of extubation outcomes were investigated as a single parameter. However, single parameters have a limited ability to predict the extubation outcome. Moreover, available data regarding the performance and cutoff values related to the previously mentioned parameters in pediatric patients are limited. Recently, a predictive model was introduced to enhance the ability of personalized factors to predict outcomes. However, the predictive model for extubation outcomes has been studied in preterm newborns and adults , 14 but 2.This prospective observational study was conducted at the pediatric intensive care unit (PICU) of a single referral university-affiliated center in Southern Thailand. Children aged 1 month to 15 years who were mechanically ventilated for more than 12 h in the PICU between January 2021 and April 2022 were enrolled. Patients with large endotracheal tube leakage (>50%) were excluded because of invalid exhalation ventilator parameters. This study was approved by the institutional review board of Faculty Medicine, Prince of Songkla University. Informed consent was obtained from the patients’ parents or guardians when the patient's clinical condition was stable and before the weaning process.2.1.2O, minimal PS based on endotracheal tube size (2) < 0.6. If patients could not tolerate the SBT, the attending physician adjusted the ventilator setting to higher than that of the minimal-setting SBT, classified as nonminimal-setting SBT. After quiet breathing was achieved, the ventilator variables consisted of P0.1, exhaled Vt (Vte), RR, PS, PEEP, and FiO2. Delta Vte was defined by the change in Vte from the initiation of SBT to 120 min after SBT to measure the decrease in tidal volume after performing SBT. Patient parameters such as oxygen saturation and vital signs were recorded at 0, 30, and 120 min and right before extubation. All ventilator variables were derived using Servo-I™ version 8.0. In addition, the corrected rapid shallow breathing index (cRSBI) was calculated by dividing the RR by Vte per the patient's ideal body weight. The standard protocol of our institutional extubation guidelines was applied to all patients to ensure good preparation and proper management . Thereafter, the attending physician adjusted the ventilator parameters according to the patient's status. Minimal-setting spontaneous breathing trial (SBT) was defined as patients receiving PS with positive end-expiratory pressure (PEEP) 3–5 cmHube size , and franagement . All dat2.2.2 in a noninvasive ventilator (NIV) or heated humidified high-flow nasal cannula (HFNC) and extubation failure. Extubation failure was defined as a requirement for reintubation. Patients with uneventful extubation were classified into the liberation respiratory support group. The cause of escalation support was recorded and patients who required escalation of respiratory support, due to stridor and respiratory distress, were classified into the upper airway obstruction (UAO) group for the subgroup analysis.Extubation outcome was assessed 48 h after extubation. Respiratory support escalation was indicated by an increasing amount of oxygen flow or FiO2.3.t-test as well as the rank sum test. In addition, multivariate analyses were conducted to identify potential factors associated with uneventful extubation. Variables with a P value of <0.2 were included in the multivariate model. Variable selection for each multivariate model was based on backward elimination. A P value <0.05 was considered statistically significant. The optimal cutoff points for P0.1, cRSBI, and Vt were defined according to the Youden method. The area under the receiver operating characteristic curve and the calculated area under the curve (AUC) were used to determine the discriminative ability of the cutoff points. A predictive scoring system for the probability of respiratory support escalation was developed using a nomogram. The model was validated using the bootstrap resampling method.The R program  was used for data analysis. Categorical variables were presented as frequencies and percentages and were compared using either the chi-square test or Fisher's exact test. Nonparametric continuous variables were presented as median [interquartile range (IQR)] and were investigated using the dependent 3.In this study, 188 mechanically ventilated pediatric patients were enrolled. The median age at the time of PICU admission and ventilator days was 1.7 years  and 3 days , respectively. The predominant indication for intubation was postoperative status (45.7%), followed by pneumonia (15.4%) and congestive heart failure (12.2%) . Forty-fP = 0.004]. Significant differences in ventilator parameters before extubation between the groups are shown in There was no difference in patient characteristics and underlying diseases between the respiratory support escalation and liberation groups . The sup3.1.P = 0.06). The optimal cutoff point of P0.1 at 30 min to predict support escalation was determined to be >0.9, with a sensitivity of 69%, specificity of 52%, and an AUC of 0.59. The optimal cutoff point for cRSBI at the beginning of the SBT was 7 breaths/min/ml/kg, with a sensitivity of 69%, specificity of 29%, and AUC of 0.54. The delta Vte change of <0 ml between 120 and 0 min had a sensitivity of 38%, specificity of 46%, and an AUC of 0.61. Patients with UAO after extubation were excluded from the subgroup analysis. The overall performance of the remaining parameters was similar to that of the entire study population   was found to be a strong predictor . P0.1 wa = 0.06) . This coRSBI is the ratio between RR and Vt (RR/Vt) and has been widely used in the adult population. The modification of the cRSBI, which is based on the patient's weight, was adjusted to suit the pediatric population. The threshold cutoff point of cRSBI at the time of SBT initiation was 7 breaths/min/ml/kg, which was comparable to the cutoff point found in a previous study . To our Although protocol-based weaning is well-established in adults, no pediatric studies have shown a clear superiority of the protocol-based approach over the physician's individualized decision . Earlier2O, and Vte/kg at 120 min of ≤8 ml/kg. This predictive model showed a superior ability, compared with single parameters, and a moderate level of diagnostic accuracy  for prognosticate escalation of care. The probability of escalating respiratory support according to this model could prove influential in optimizing patient care, for example, preparing and closely monitoring at-risk patients.Although multiple predictors were analyzed in this study, the accuracy of each weaning indicator exhibited a limited ability to predict escalation of respiratory support. Presently, predictive models utilizing combined parameters are increasing in popularity as tools of choice for both diagnosis and prognosis. However, no other study has explored this predictive model in a pediatric population. In the present study, the predictive model consisted of four feasible bedside indexes: nonminimal-setting SBT, >3 ventilator days, P0.1 at 30 min of ≥0.9 cmHThis study has some limitations. As the majority of the study population consisted of postoperative patients, the extubation failure rate was low. Moreover, parameters associated with UAO after extubation were not explored. However, after excluding patients with UAO, the subgroup analysis revealed unchanged outcomes. Furthermore, inhomogeneous indications for intubation could have affected the study outcomes. In light of these limitations, the generalizability of our study's results requires careful consideration. A large prospective study investigating the performance of predictive parameters is warranted. Moreover, the proposed predictive scoring system requires external validation.5.2O at 30 min after SBT, corrected RSBI of >7 breaths/min/ml/kg, decreased Vt over 120 min of SBT, >3 ventilator days, and nonminimal-setting SBT were found to be risks for respiratory support escalation after extubation. The evaluated predictive model, which utilized parameters in combination, performed best when defining the probability of the need for support escalation after extubation.A P0.1 value of >0.9 cmH


Intestinal lymphangiectasia (IL) is a rare protein-losing enteropathy caused by disorders of the intestinal lymphatics. There are only a few case reports and case series concerning the VCE (video capsule endoscopy) findings of IL. This work aimed to evaluate the VCE characteristics of small intestinal mucosal abnormalities in patients with IL, and to investigate the relationship between clinical and VCE characteristics.Consecutive patients with IL who underwent VCE were enrolled in this retrospective study. The cases were classified into the white villi group and non-white villi group according to mucosal abnormalities detected by VCE. Clinical and endoscopic characteristics were investigated and analyzed.p = 0.008). In VCE, there were no significant differences in the involved segments and total detected rate of complications between the white villi and non-white villi groups (p > 0.05), while the detected rate of lymphatic leakage in the white villi group was significantly higher than that in the non-white villi group .A total of 98 patients with IL with a median onset age of 26.3 ± 19.2 years were included. VCE revealed the following small intestinal lesions: (i) white villi type , i.e.: white-tipped or granular villi, white nodular villi or plaques; (ii) non-white villi type , i.e.: diffused low and round villi; (iii) complications , i.e.: bleeding, ulcers, protruding or vesicular-shaped lesions, stenosis and lymphatic leakage. A total of 58.2% (57) and 41.8% (41) of the cases were classified into the white villi and non-white villi groups respectively. The percentage of chylothorax in the white villi group was significantly lower than that in the non-white villi group (12/57 vs. 19/41, Our study evaluated the entire small intestinal mucosal abnormalities of IL by VCE, especially endoscopic complications. IL has specific VCE abnormalities in addition to classical endoscopic findings. Intestinal lymphangiectasia (IL) is a rare protein-losing enteropathy (PLE) caused by disorders of the intestinal lymphatics . It is cAccording to the etiology, IL can be classified into primary and secondary IL. Primary intestinal lymphangiectasia (PIL) is considered a primary congenital disorder , whose pEndoscopy is one of the main modalities for the diagnosis of IL. However, conventional esophagogastroduodenoscopy (EGD) and colonoscopy cannot visualize the entire intestine. Single balloon or double balloon enteroscopy is invasive and cannot ensure the complete evaluation of the entire small intestine. Video capsule endoscopy (VCE), a novel device introduced in the last decade, could be used to explore the whole segment of the small intestine , 9 and iTo the best of our knowledge, there are only a few case reports and case series concerning the VCE findings of IL –15. In tThis retrospective study was implemented at the Digestive Endoscopy Center and Department of Lymphatic Surgery, Beijing Shijitan Hospital, Capital Medical University, China, from January 2011 to December 2021. Consecutive patients with IL who underwent VCE were enrolled in the study. Patients with pacemakers, pregnant, breastfeeding, those with incomplete clinical data or lost to follow-up were excluded.Data from the following variables was collected: age, sex, main complaint, medical history, biochemical test, radiological results, VCE findings and treatment.All patients or legal guardians of the patients gave informed consent before VCE. This study was approved by the Research Ethics Committee of the Beijing Shijitan Hospital, Capital Medical University (sjtkyll-lx-2022074). All the data were anonymously collected and analyzed.The diagnosis of IL was suspected in patients with the evidence of PLE and confirmed by the presence of typical endoscopic findings or pathologically confirmed dilation of lymphatic lacteals in endoscopic biopsy or surgical specimens. PLE was confirmed by technetium-99 m human serum albumin (HSA) scintigraphy, accompanied by hypoproteinemia  –18.According to the etiology, IL was classified into PIL and SIL .VCE  was used. All patients followed a clear liquid diet for 24 h with fasting for 12 h prior to the examination. Polyethylene glycol  or oral electrolyte solution  was used for the bowel preparation. Fasting patients swallowed a capsule with a glass of water, 2 h after which they were allowed to drink or eat. VCE recording lasted 8–16 h. Video data stored on a portable device was then transferred to a computer to be analyzed by RAPID Reader® 7/8 software (Given Imaging). Images were displayed with two windows at 10 times speed in manual mode and reviewed by two endoscopists independently (Lin Lin and Kuiliang Liu). The consensus was reached after discussion if the opinion was not consistent. During the review, the entire small intestine was divided into three equal parts  according to the transit interval of the capsule in the small intestine and then evaluated individually according to the method described by Goldstein et al. and Gralnek et al. .In this study, the cases were classified into the white villi group and non-white villi group according to mucosal abnormalities detected by VCE. A flow diagram showing the different groups of patients with IL is described in Fig. According to Ohmiya N et al., endoscopic mucosal abnormalities were classified into the white villi type and non-white villi type . The whiComplications detected by VCE were recorded as bleeding, ulcers, protruding or vesicular-shaped lesions, stenosis and lymphatic leakage. Lymphatic leakage was defined as whitish chyle detected in the intestinal lumen.2) test or Fisher’s exact test. Continuous variables were expressed as the means ± standard deviation (SD) and range and compared using the Student’s t test. All p values were two-tailed and the level of statistical significance was set at 0.05.The data was analyzed using Statistical Product and Service Solution version 23.0 . Categorical variables were expressed as numbers and percentages (%) and compared using the chi-square  and 45 females (45.9%), with a median age at diagnosis of 32.0 ± 18.7 years (range: 4–72 years) and median age at onset of 26.3 ± 19.2 years (range: 0–68 years) were included. The median duration of disease from the onset of symptoms to diagnosis was 5.7 ± 6.8 years (range: 0.1–44 years). The onset age of 35 patients (35.7%) was lower than 18 years.Among them, 68 patients (69.4%) were diagnosed with PIL, and 30 patients (30.6%) were diagnosed with SIL. Secondary epidemic factors include portal hypertension (n = 15), chronic heart failure (n = 7), systemic lupus erythematosus (n = 2), thrombophilia (n = 4), and cryptogenic multifocal ulcerous stenosing enteritis (n = 2). The main complaints at onset included edema , diarrhea , abdominal distension , fatigue  and abdominal pain . In addition, 31 patients (31.6%) suffered from chylothorax, and 52 patients (53.1%) suffered from chylous ascites.All patients were advised for low-fat diet supplemented with medium chain triglycerides (MCT). Thoracic duct surgery to reduce lymphatic pressure was performed on 86 patients (87.8%); among them, other surgical methods, such as exploratory laparotomy and partial enterectomy, were performed on 15 patients.VCE revealed the following small intestinal lesions: (i) white villi type , i.e.: white-tipped or granular villi, white nodular villi or plaques Fig. .Fig. 2ViA total of 96.9% (95/98), 65.3% (64/98) and 49% (48/98) of the cases had the small intestinal abnormalities involving the proximal 1/3, middle 1/3, and distal 1/3 segments, respectively.p = 0.006), while the mean duration before diagnosis in the white villi group was significantly longer than that in the non-white villi group . More cases of SIL were detected in the non-white villi group than that in the white villi group . The percentage of chylothorax in the white villi group was significantly lower than that in the non-white villi group . The levels of triglyceride and immunoglobulin G in the white villi group were significantly higher than those in the non-white villi group, while the level of complement 3 was significantly lower (p < 0.05). There were no significant differences in main complaints, chylous ascites, the levels of lymphocytes, hemoglobin, serum albumin, serum globulin, or C-reactive protein (p > 0.05).As shown in Table As shown in Table p > 0.05), but the detected rate of lymphatic leakage in the white villi group was significantly higher than that in the non-white villi group .There was no significant difference in the total detected rate of complications in the different groups . The onset age of 35 patients (35.7%) was lower than 18 years. Before the definite diagnosis, the duration time varied. A total of 69.4% of the cases were diagnosed as PIL, and secondary etiologies were found in 30.6% of the cases. PIL is considered congenital in origin, most likely caused by failure in the proper formation of lymphatic vessels during infancy or earlier, and mutation or dysfunction of some genes or transcriptional factors, such as vascular endothelial growth factor-C, VEGFR3, prosperorelated homeobox-transcriptional factor, forkhead transcriptional factor and SOX18, have been reported to be related to lymphedematous diseases and lymphangiectasia –22. IL cTypical findings of dilated lymphatics, such as pinhead-sized, white-yellow lesions can usually be detected in the second part of the duodenum by EGD in some IL cases , 26. AbnThe present endoscopic classifications of IL were always based on EGD, colonoscopy, intraoperative enteroscopy and via enteroscopy , 28. A fIn addition, some complications were described by VCE, such as bleeding, ulcers, protruding or vesicular-shaped lesions, stenosis and lymphatic leakage. Nodular protrusion without whitish mucosa has been described as one kind of endoscopic finding of IL , 30. In One of the major complications of IL is lymphatic leakage causing hypoproteinemia and malnutrition. Traditionally, 99mTc-DX lymphatic imaging and direct lymphangiography can be used to detect lymphatic leakage according to the presence of radioactivity leaking out of lymphatics and into the intestine , 31. SomNutritional therapy comprising a high-protein, low-fat diet, supplemented with MCT is the simplest, most effective, and widely prescribed treatment with the fewest side effects , 38. OctThis study summarized the clinical and endoscopic characteristics of IL, and highlighted the usefulness of VCE in the entire small intestinal mucosal abnormalities. Although IL is a rare disease, a total of 98 cases were enrolled in our study, the largest number using VCE to assess IL to date. It is ideal if the appropriate strategy of treatments and surveillance could be formulated according to the endoscopic classification. More investigation is required in the future to evaluate the clinical relationship of the endoscopic abnormalities.Our study evaluated the entire small intestinal mucosal abnormalities of IL by VCE, especially endoscopic complications. Clinical characteristics vary between white villi type and non-white villi type patients with IL. IL has specific VCE abnormalities in addition to classical endoscopic findings.


The management of periprosthetic joint infections (PJI) of the lower limb is challenging, and evidence-based recommendations are lacking. The present clinical investigation characterized the pathogens diagnosed in patients who underwent revision surgery for  PJI of total hip arthroplasty (THA) and total knee arthroplasty (TKA).The present study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The institutional databases of the RWTH University Medical Centre of Aachen, Germany, were accessed. The OPS (operation and procedure codes) 5–823 and 5–821 and the ICD  codes T84.5, T84.7 or T84.8 were used. All patients with PJI of a previous THA and TKA who underwent revision surgery were retrieved and included for analysis.Data from 346 patients were collected (181 THAs and 165 TKAs). 44% (152 of 346 patients) were women. Overall, the mean age at operation was 67.8 years, and the mean BMI was 29.2 kg/m2. The mean hospitalization length was 23.5 days. 38% (132 of 346) of patients presented a recurrent infection.PJI remain a frequent cause for revisions after total hip and knee arthroplasty. Preoperative synovial fluid aspiration was positive in 37%, intraoperative microbiology was positive in 85%, and bacteraemia was present in 17% of patients. Septic shock was the major cause of in-hospital mortality. The most common cultured pathogens were Staph. epidermidis, Staph. aureus, Enterococcus faecalis, and Methicillin-resistant Staph aureus (MRSA). An improved understanding of PJI pathogens is important to plan treatment strategies and guide the choice of empirical antibiotic regimens in patients presenting with septic THAs and TKAs.Level of Evidence: Level III, retrospective cohort study. Arthroplasty aims to restore quality of life in patients with end-stage joint degeneration, fractures, or joint infections , 2. AccoThe present study was conducted according to the principles of the Declaration of Helsinki and was approved by the ethics committee of the RWTH Aachen University (project ID EK 121/22). The present study follows the Strengthening the Reporting of Observational Studies in Epidemiology: the STROBE Statement . The preAll patients with a PJI of THA or TKA who had undergone THA or TKA revision surgery were retrieved. The inclusion criteria were: arthroplasty of knee or hip; microbiological evidence of pathogen using joint aspiration and/ or intraoperative histologic examination and/ or of blood cultures; the presence of at least one of these signs of inflammation at the joint: heat , pain (dolor), redness (rubor), and swelling (tumor). The exclusion criteria were: any other non-infective ailment in a previously implanted arthroplasty; arthroplasty performed in joints other than knee and hip.The following data were recorded: gender, age at admission, height, weight and BMI, side, joint and the year of implantation. Data concerning the number and length of hospitalisation and the number of revisions were collected. Information on the type of pathogen was collected. Data on mortality were also retrieved. The perioperative risk was assessed using the American Society of Anaesthesiologists (ASA) score .All analyses were conducted by the main author (FM) using the IBM SPSS Statistics software package, version 25. For descriptive statistics, frequency (amount of events/ number of observations) was used for binary variables. Arithmetic mean and standard deviation were adopted for continuous variables.N = 474), not performed at the knee or hip (N = 231), no evidence of infection (N = 209), no data of patients available (N = 64), uncertain data (N = 7). Finally, 346 patients were considered in the present study  of patients presented a recurrent infection. Demographic information of the patients is shown in Table Data from 346 patients were collected (181 THAs and 165 TKAs). 44% (152 of 346 patients) were women. Overall, the mean age was 67.8 years, the mean BMI 29.2 kg/mIn the 346 patients, a maximum of 18 operations were performed per in-hospital stay. In three patients, no operation was performed. On average, 2.6 ± 0.79 revisions per patient were performed. Overall, patients' surgical procedures lasted a mean of 213 ± 183.8 min. The shortest surgical duration was 36 min . The longest surgical duration was 1112 min . Liners were changed in 171 patients; the implants were removed in 155 cases. Amputation was necessary for two patients. Pathogens were detected in 37% (128 of 346) of joint aspirations, 85% (294 of 346) of intraoperative microbiologic examinations, and 17% (59 of 346) of blood cultures. 312 (90%) patients survived, and 34 (10%) patients died during inpatient stay. 31 of these 34 (90%) died from septic shock. One patient died of kidney failure, one of ventricular fibrillation, and one of small cell lung cancer progression . Considering the high prevalence of diabetics in the present study, diabetes mellitus appears to be a relevant comorbidity for the clinical course [However, the influence of enterococcal infections on mortality was not significant compared to other pathogens. In addition, l course .Periprosthetic joint infections remain a frequent cause for revisions after total hip and knee arthroplasty. Preoperative synovial fluid aspiration was positive in 37% of patients, intraoperative microbiology was positive in 85% and bacteraemia was present in 17%. Septic shock was the major cause of in-hospital mortality. The most common cultured pathogens were Staph. epidermidis, Staph. aureus, Enterococcus faecalis, and Methicillin-resistant Staph. aureus (MRSA). An improved understanding of PJI pathogens is important to determine treatment strategies and guide the choice of empirical antibiotic regimens in patients presenting with septic THAs and TKAs.


Current guidelines recommend potent P2Y12 inhibitors for patients after acute coronary syndrome. However, the data on the efficacy and safety of potent P2Y12 inhibitors in elderly Asian populations was limited. We aimed to investigate the major adverse cardiovascular events (MACE), bleeding events, and net adverse clinical events (NACE) with ticagrelor and clopidogrel in Taiwanese patients aged 65 and older after acute myocardial infarction (AMI).This retrospective population-based cohort study was conducted using data from the National Health Insurance Research Database. The AMI patients aged ≥65 years who underwent percutaneous coronary intervention (PCI) and survived after 1 month were included. The patients were separated into 2 cohorts depending on the type of dual antiplatelet therapy (DAPT) they received: ticagrelor plus aspirin (T + A) or clopidogrel plus aspirin (C + A). We used inverse probability of treatment weighting to balance the difference between these 2 study groups. The outcome included all-cause mortality, MACE , intracerebral hemorrhage, major bleeding, and NACE which is composed of cardiovascular death, ischemic and hemorrhagic events. The follow-up period was up to 12 months.p = 0.006] and 0.58 , respectively. No differences were found in MACE, intracranial and major bleeding between the 2 groups. In addition, the patients with T + A had a lower risk of NACE with an adjusted HR of 0.86 From 2013 to 2017, a total of 14,715 patients who met the eligibility criteria were separated into 2 groups: 5,051 for T + A and 9,664 for C + A. Compared to patients with C + A, patients who received T + A had a lower risk of cardiovascular death and all-cause death, with an adjusted HR of 0.57 [95% confidence interval (CI), 0.38–0.85, Among elderly AMI patients receiving DAPT after successful PCI, ticagrelor was a more favorable P2Y12 inhibitor than clopidogrel because of lowering the risk of death and NACE without increasing the risk of severe bleeding. Ticagrelor is an effective and safe P2Y12 inhibitor in Asian elderly survivors after PCI. The Asia-Pacific region is populated by more than 4.2 billion inhabitants, equivalent to 60% of the world's population. Acute coronary syndrome (ACS) is now a major cause of death and disability in this region as the Western conturies . Dual anIn the global randomized controlled Phase III PLATO trial, ticagrelor, a more potent P2Y12 receptor antagonist, has been demonstrated to reduce the composite end-point of myocardial infarction (MI), stroke, and death from cardiovascular causes across different age groups . HoweverThis retrospective population-based cohort study was conducted by using data from the National Health Insurance Research Database (NHIRD) that was maintained by the Health and Welfare Data Science Center, Ministry of Health and Welfare. The NHIRD was a claims-based database that covered healthcare utilization and costs for 99% of residents in Taiwan. The NHIRD files included inpatient, outpatient, and pharmaceutical claims. The data of disease diagnosis was coded by the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM), and the Tenth Version (ICD-10-CM) after 2016. The data of prescription drugs can be classified based on Anatomical Therapeutic Chemical (ATC) classification. In addition, the enrollment files of beneficiaries and providers were included. The data period used in this study was from 2013 to 2017. The study protocol had been reviewed and approved by Taipei Medical University Joint Institutional Review Board (N202205097).Patients first diagnosed with AMI, including ST elevation (STEMI) and non-ST elevation myocardial infarction (NSTEMI), and who underwent PCI between 2013 and 2017 were included. The period was chosen to follow the regulations of the National Health Insurance Administration because ticagrelor was first reimbursed for treating patients with AMI after July 1st, 2013. The reimbursed DAPT duration was 9 months after AMI. Of these patients, we added several exclusion criteria to ensure that the patients were eligible for the current study, including (1) age less than 65 years, sex information missing, or not a resident in Taiwan; (2) did not have heparin or antiplatelet agents during the AMI admission; (3) had anticoagulant drugs or had aspirin only during the AMI admission; (4) died within a month after the index AMI; and (5) had a history of coronary artery bypass graft during the study period. Of the patients, we excluded those who had no prescription claim of DAPT, switched the type of DAPT, or had a medication possession ratio (MPR) <0.8 within a month after AMI discharge. MPR is defined as the total number of days covered by filled prescriptions divided by a predefined period . The value of MPR ≥0.8 suggests a significantly higher adherence . Figure 2DS2-VASC score  for quantifying thromboembolic risk , and HAS-BLED score  were used , cardiovascular death, and all-cause death to evaluate the effectiveness of therapy. MACE is defined as a composite of nonfatal ischemic stroke nonfatal MI, and cardiovascular death. The events of intracerebral hemorrhage (ICH) and major bleeding were considered to assess the safety of the treatment. The definition of major bleeding was hospitalized due to bleeding, symptomatic bleeding in a critical area or organ, or bleeding leading to transfusion of more than 2 units of packed red blood cells. We also analyzed the net adverse clinical events (NACE), composed of cardiovascular death, ischemic events , and hemorrhagic events . The follow-up period was up to 12 months. The disease diagnosis codes for study outcomes are provided in p value <0.05 was considered significant.Baseline characteristics were analyzed using standardized mean difference. A standardized mean difference of greater than 0.1 indicates the non-negligible difference between the two groups. Inverse probability of treatment weighted (IPTW) analysis was conducted to adjust for differences in baseline characteristics between the two cohorts. Cox proportional hazard regression adjusted for baseline covariates and medications listed in Among 52,692 patients first diagnosed with AMI and underwent PCI between 2013 and 2017, a total of 14,715 patients, including STEMI (49.7%) and NSTEMI (50.3%) met the eligibility criteria. The patients were separated into two cohorts depending on the type of DAPT: 5,051 for ticagrelor plus aspirin (T + A) and 9,664 for clopidogrel plus aspirin (C + A) .2DS2-VASc, ORBIT, and HAS-BLED scores. Also, more patients had received T + A in more recent years. The mean (±standard deviation) DAPT duration of T + A and C + A was 7.1 (±3.9) and 7.9 (±3.7) months (p = 0.218), respectively. After IPTW, baseline characteristics were well-balanced between the 2 groups.Baseline characteristics are shown in p = 0.006) and 0.58 . The MACE events rate was lower in the T + A group, although the p-value was insignificant. The recurrent MI and ischemic stroke events rate were comparable in 2 groups  while no different in age groups.The incidence and relative risks of ICH and major bleeding are shown in This nationwide population-based cohort study found that ticagrelor was superior to clopidogrel in lowering the risk of death and NACE while not increasing the risk of marjor bleeding among elderly AMI patients after PCI. The phenomenon was found in those aged 65 years and older.Studies have shown that compared to clopidogrel, the more potent P2Y12 inhibitors, including prasugrel and ticagrelor, further reduced the risk of death, MI, and stroke in ACS patients , 19. ButThe fear of increased bleeding risk and the intrinsic frailty of this aging population often lead physicians to prescribe a less potent P2Y12 inhibitor. As a result, those patients might be potentially undertreated and exposed to a higher incidence of thrombotic complications –26. AccoThis current study confirmed the significant survival benefit of ticagrelor over clopidogrel and similar major bleeding risk between treatment groups. The similar findings were observed in the PLATO trial although a recent randomized open-label study, POPular-AGE study, including patients aged ≥70 years with non-ST elevation ACS showed similar thrombotic events between ticagrelor and clopidogrel but less bleeding in the clopidogrel group . The posp = 0.001). These pleomorphic effects of ticagrelor beyond what we know about the platelet inhibition might provide some hypothesis for our study's survival benefit of ticagrelor over clopidogrel (Meanwhile, ticagrelor might have pleiotropic effects that go beyond antiplatelet action. A recent study has indicated that ticagrelor may have antimicrobial activity in a mouse model . But, onpidogrel , 35. FurAlthough short duration or de-escalated DAPT strategy might reduce bleeding without increasing cardiovascular events in some studies, but current guideline still recommend DAPT consisting of a potent P2Y12 receptor inhibitor in addition to aspirin for 12 months in acute coronary syndrome patients after PCI, irrespective of the stent type, unless there are contraindications adequate –38. StudThere were some limitations in this study. First, the NHIRD does not have certain patient information, such as risk behaviors, diet, and physical activities, which might be associated with the outcomes of interest. Second, the NHIRD does not contain clinical information, such as angiographic findings during PCI, the extent of coronary artery disease, and the severity of AMI at admission. Consequently, we could not adjust for the severity of AMI nor could we know whether the AMI admission was planned or not, which might induce non-differential misclassification bias. We did not follow BARC or GUSTO, or TIMI criteria for bleeding classification because of the limitation of NHIRD that laboratory data like the hemoglobin or other exact clinical records was unable to obtain. Therefore, some outcome measurement like minor bleeding might be underestimated. In this study, we defined the major bleeding as being hospitalized due to bleeding, bleeding in a critical organ, or bleeding leading to transfusion that were comparable to the major bleeding of TIMI or BARC 2–3 bleeding criteria. The similar method has been used in the claim-based studies , 39. ThiUsing the nationwide cohort study, we found that in Asian patients aged 65 years or older presenting with AMI, ticagrelor provided a more favorable outcome than clopidogrel, because it was associated with a lower cumulative rate of all-cause mortality, cardiovascular death and NACE without an increase of intracranial or major bleeding.