Patients and Methods. A retrospective survey was used to collect clinical data of patients undergoing percutaneous coronary intervention due to CAD, and CKD patients were screened for further analysis. According to whether patients had received coronary rehabilitation treatment, research subjects were divided into two groups, the coronary rehabilitation group and the noncoronary rehabilitation group. The baseline characteristics of the propensity score matching between the two groups were compared. Survival analysis used the Cox hazard ratio (HR) model as regression method to compare the relative risk of the endpoints in the coronary rehabilitation group and the noncoronary rehabilitation group.  According to researches, many people with chronic kidney disease (CKD) had the higher incidence rate and mortality rate of coronary artery disease (CAD) after percutaneous coronary intervention than those who did not receive percutaneous coronary intervention, while coronary rehabilitation was beneficial for patients who received percutaneous coronary intervention I. This study aims to analyze whether coronary rehabilitation was beneficial to patients with CKD after percutaneous coronary intervention. P > 0.05). CKD patients receiving coronary rehabilitation had a significant reduction in all-cause mortality  and cardiac complications . Survival analysis showed that the survival rate of the coronary rehabilitation group was significantly higher than that of the noncoronary rehabilitation group (P=0.024).  From January 2007 to January 2012, a total of 246 CKD patients treated with percutaneous coronary intervention were included in this study, and 106 of them obtained coronary rehabilitation after surgery. After propensity score matching, there were 89 pairs of patients in the two groups who had no significant difference in demographic and clinical characteristics (all  For CKD patients undergoing percutaneous coronary intervention, receiving cardiac rehabilitation can significantly improve long-term survival and reduce cardiac events. Although the mortality rate of people suffering from coronary artery disease (CAD) has been on a downward trend due to the successful development of more and more effective interventional technologies and cardiovascular drugs –3, serioChronic kidney disease (CKD) complication of CAD has attracted social attention for the increasing incidence of CAD and the long-term risks of cardiac events , 11. Mor2; (3) the patient who had no contraindications of coronary rehabilitation including unstable angina, severe heart failure (with LVEF < 30%), and uncontrolled ventricular arrhythmia [We retrospectively compiled the clinical data of patients who meet the following criteria: (1) the patient diagnosed with ACS and receiving percutaneous coronary intervention treatment according to the 2011 ACCF/AHA/SCAI guideline ; (2) therhythmia ; (4) theThe study compared demographical characteristics, examination results during hospitalization, and long-term subsequent prognosis outcomes. The study was also approved by the hospital Medical Ethics Committee.Accorded to the abovementioned criteria, there were 246 eligible CKD patients receiving percutaneous coronary intervention in the hospital between January 2007 and January 2012. Accorded to whether the patients had received coronary rehabilitation treatment, the research subjects were divided into two groups, the coronary rehabilitation group and the noncoronary rehabilitation group.15, the coronary rehabilitation program was launched in the Department of Cardiology Hospital, since February 2010, when a group of coronary rehabilitation was founded including cardiologists, nurse specialists, physiotherapists, dietitians, psychologists, exercise specialists, occupational therapists, and clerical administrators. A well-designed coronary rehabilitation plan was made for each patient who was willing to receive treatment. The coronary rehabilitation plan included the following steps: first of all, health education and habit correction; secondly, lifestyle analysis and risk factor control ; third, psychological counseling and evaluation; fourth, medical evaluation and risk factor control; fifth, cardioprotective therapies; sixth, long-term follow-up on time; and seventh, regularly evaluate the coronary rehabilitation program. The patients who received coronary rehabilitation plan needed to attend the coronary rehabilitation sessions within 3 months after percutaneous coronary intervention and the multidisciplinary program must last more than 1 year (more than 3 sessions of coronary rehabilitation).According to the BACPR Standard 2The follow-up lasts from January 2010 to January 2017 by telephone or out-patient clinics; each patient was given an annual inquiry of their conditions. The data were regarded as meaningless if the patient's clinical data were incomplete or the contact was interrupted during long-term follow-up. Primary and secondary endpoint studies were as follows: the primary endpoint was defined as all-cause mortality during long-term follow-up; the secondary endpoints were defined as composite adverse outcomes, including adverse outcomes such as myocardial infarction and unexpected revascularization and so on.In this study, IBM SPSS Statistics version 19.0  was used for statistical analysis. Firstly, the normal distribution test of variables was carried out to check the distribution of variables. Continuous variables conforming to normal distribution were expressed as mean ± standard deviation, and classified variables were expressed as proportion.t-test. The chi-square test was performed in different evaluations of categorical variables. Multiple logistic regression was performed to calculate propensity scores by considering demographic and clinical variables. Use the “greedy matching” method to match patients with the closest propensity score. After propensity score matching, Student's t-test and McNemar paired sample were used for analysis.The comparison of continuous variables between the two groups used the independent sample P value less than 0.05 was considered as statistically significance.Survival analysis used Cox hazard ratio (HR) model as a regression method to compare the relative risk of the endpoints in the coronary rehabilitation group and the noncoronary rehabilitation group. We performed univariate analysis between covariates and endpoints. The Kaplan–Meier survival curve and log rank test were used to compare the survival status of the coronary rehabilitation group and the noncoronary rehabilitation group. P=0.001), and the BMI of the coronary rehabilitation group was lower than that of the noncoronary rehabilitation group (P=0.003). There were also significant differences in the time between the most recent MI (P=0.011) and the percutaneous coronary intervention type in the same period (P=0.001). As for follow-up treatment, more patients in the coronary rehabilitation group received drug-eluting stent treatment (P=0.007). In terms of comorbidities, the coronary rehabilitation group had a higher proportion of heart failure (P=0.004) and hypertension (P=0.015). There were no significant differences on gender and smoking status (P > 0.05). There were no statistical differences in the proportion of diabetes, chronic lung disease, cerebrovascular disease, and tumor (P > 0.05). After the propensity match, there were no statistical differences in the variables listed in P > 0.05).There were 246 eligible research subjects that were included in this study. 106 patients were enrolled in the coronary rehabilitation group and 140 patients in the noncoronary rehabilitation group. The demographical characteristics and clinical data of the two groups are shown in P=0.029). Survival analysis also showed that the survival rate of the coronary rehabilitation group was higher than that of the noncoronary rehabilitation group (P=0.024).During the average (38.9 ± 22.5) months of follow-up, there were 37 primary endpoint events , including 28 cardiovascular deaths and 9 noncardiovascular deaths. As shown in P=0.044). There were no statistical significance in subgroup analysis of myocardial infarction and emergency revascularization (P > 0.05). The Kaplan–Meier survival curve showed that the coronary rehabilitation group had fewer cardiovascular events than the noncoronary rehabilitation group (O = 0.039).During the follow-up period, 44 secondary endpoint events occurred, including 18 cases of myocardial infarction and 26 cases of emergency revascularization. Compared with the noncoronary rehabilitation group, the risk of cardiac events in the coronary rehabilitation group was significantly lower, with HR of 0.532 95% CI (0.287–0.984) (As far as we know, this study retrospectively compared patients treated with coronary rehabilitation and patients who did not receive coronary rehabilitation. It was the first time that coronary rehabilitation can effectively reduce all-cause mortality and cardiac events in patients with CKD after percutaneous coronary intervention. It is worth mentioning that, in this study, the conclusions were more convincing as baseline bias was eliminated by propensity score matching.Previous studies had provided a close link between coronary rehabilitation training and the reduction of all-cause mortality . A systeCKD is an independent risk factor for CAD. CAD is the main cause of incidence rate and mortality of CKD patients –22. For Univariate Cox regression analysis was performed between the two groups to compare the all-cause and mortality and cardiac events in the coronary rehabilitation group and the noncoronary rehabilitation group. The result was positive which was caused by two main mechanisms. On the one hand, coronary rehabilitation complexes including exercise, smoking cessation, and weight loss have been shown to be beneficial to endothelial function, which may play a role in improving renal function and revascularization of coronary arteries . On the No matter how great the benefits of coronary rehabilitation are, without people participation and compliance, the result is nonsense. Despite years of promotion, the participation rate of coronary rehabilitation is still very low, especially in countries with undeveloped medical conditions. According to the Canadian Institutes of Health Research, only 38.8% of countries have heart rehabilitation programs, of which 68.0% are in high income countries and 23% are in low and middle income countries . In ChinIt should also be noted that there are several limitations of this study. Firstly, retrospective design has effect on the strength of evidence. Secondly, the follow-up data are not as good as the cohort study, the proportion of censored data is relatively high, and there may be a certain deviation in the final result. Finally, because the clinical data of CKD are incomplete, this study lacked CKD staging information.In conclusion, this study demonstrated that long-term coronary rehabilitation program would effectively reduce the risk of death and the incidence of cardiac events in patients with CKD. More efforts are needed to promote coronary rehabilitation in patients with CAD, especially for those with CKD.


Currently utilized antidepressants have limited effectiveness and frequently incur undesired effects. Most antidepressants are thought to act via the inhibition of monoamine reuptake; however, direct binding to monoaminergic receptors has been proposed to contribute to both their clinical effectiveness and their side effects, or lack thereof. Among the target receptors of antidepressants, α1‑adrenergic receptors (ARs) have been implicated in depression etiology, antidepressant action, and side effects. However, differences in the direct effects of antidepressants on signaling from the three subtypes of α1-ARs, namely, α1A-, α1B- and α1D‑ARs, have been little explored. We utilized cell lines overexpressing α1A-, α1B- or α1D-ARs to investigate the effects of the antidepressants imipramine (IMI), desipramine (DMI), mianserin (MIA), reboxetine (REB), citalopram (CIT) and fluoxetine (FLU) on noradrenaline-induced second messenger generation by those receptors. We found similar orders of inhibition at α1A-AR (IMI < DMI < CIT < MIA < REB) and α1D‑AR (IMI = DMI < CIT < MIA), while the α1B-AR subtype was the least engaged subtype and was inhibited with low potency by three drugs (MIA < IMI = DMI). In contrast to their direct antagonistic effects, prolonged incubation with IMI and DMI increased the maximal response of the α1B-AR subtype, and the CIT of both the α1A- and the α1B-ARs. Our data demonstrate a complex, subtype-specific modulation of α1-ARs by antidepressants of different groups. Depression is a debilitating disorder causing lasting suffering in millions of people, reducing their ability to live normal lives and representing a major, although often hidden, burden to society. It is estimated that more than 300 million people suffer from depression worldwide ; for exaPhysiological responses to noradrenaline and adrenaline are mediated by adrenergic receptors (ARs), which are widely distributed in the central and peripheral nervous systems. These receptors are the seven transmembrane-spanning receptors that belong to the large G-protein-coupled receptor (GPCR) superfamily. The AR family is presently divided into three distinct receptor subclasses, namely, β-, α2- and α1-ARs, and each subclass comprises several subtypes . The α1-2+. Although the three α1-AR subtypes induce the same cascade of intracellular signaling events, they differ in their subcellular distribution, efficacy in evoking intracellular signals, and transcriptional profiles .,19.2+. AAll three α1-AR subtypes are present in the peripheries of both the cardiovascular and genitourinary systems and at relatively high densities throughout the CNS. In the periphery, both the differential distribution and function of the α1A-, α1B- and α1D-AR subtypes are well characterized; for example, this knowledge was applied successfully to minimize the side effects of drugs for benign prostatic hypertrophy ,19. HoweMore recently, the utilization of genetic mouse models has supported the notion that different α1A-, α1B- and α1D-AR subtypes have not only divergent, but sometimes even opposing, roles in the regulation of behaviors linked with depression and other neuropsychiatric disorders ,19,22. TSignaling from α1A-, α1B- and α1D-ARs can be regulated at multiple levels, including transcriptional regulation, protein trafficking, and desensitization ,28. All Considering the evidence for the role of α1-ARs in depression and antidepressant action, together with recent data demonstrating the differential binding of clinically relevant antidepressants to the α1A-, α1B- and α1D-AR subtypes, the aim of the current study was to evaluate how the chosen antidepressants modulate intracellular signal transduction from α1A-, α1B- and α1D-ARs, both acutely and after prolonged treatment, by utilizing an in vitro overexpression model and the measurement of second messenger generation.Our main findings demonstrate that imipramine, desipramine and mianserin directly antagonize the noradrenaline-induced second messenger generation of all three α1-AR subtypes, albeit with different potencies, while reboxetine and citalopram directly antagonized the responses of α1A-ARs and both the α1A- and α1D-AR subtypes, respectively. Importantly, however, we show that prolonged incubation with imipramine and desipramine increases the maximal response to α1B-AR, while citalopram increases the maximal response to the α1A subtype and, to a lesser extent, the α1B subtype. Together, our results show that the interaction of various antidepressants and α1-ARs might involve both the antagonism and the modulation of the strength of intracellular signal transduction.We created two PC12 cell lines stably expressing human α1A-AR or α1B-AR via the transfection of wild-type PC12 cells with pcDNA3.1+ plasmids encoding human α1A-AR and α1B-AR and selection under antibiotics A,B. No a50 of 1 × 10−6.2 for noradrenaline and an EC50 of 1 × 10−6.1  than α1B-AR (IC50 = 1 × 10−7.5)  F. For α13H]prazosin, and in parallel, nonspecific binding was determined in the presence of a non-labeled prazosin  was relatively similar for all cells, i.e., 3622 ± 247 binding sites per cell for α1A-AR  of 0.32 ± 0.08, 0.83 ± 0.16 and 0.90 ± 0.14 nM for α1A-  receptor response (4 μM for α1A- or α1B-AR and 1 μM for α1D-AR). Then, after 75 or 95 min of incubation , the levels of second messengers generated were subsequently evaluated. We found that imipramine .The order of antidepressant potency in inhibiting α1A-AR was IMI < * DMI < * CIT < * MIA < * REB, which was similar to that of the inhibition of α1D‑AR by these drugs (IMI = DMI < * CIT < * MIA), but the tested drugs had a different order of potency in inhibitting the α1B‑AR subtype (MIA < * IMI = DMI)  for 24 h , Table 2−10 to 1 × 10−4 M, followed by the measurement of second messenger generation. We found subtype-specific differences in the response to noradrenaline after preincubation with different antidepressant drugs. Namely, imipramine increased the maximal response of α1B-AR after 24 h of preincubation  with noradrenaline at concentrations ranging from 1 × 10cubation . The effcubation ; additioeceptors . Desipraeceptors ; howeverter 24 h  but not ter 24 h . Mianserter 24 h , but sliter 24 h , and shie latter . Citalope latter  and 120 e latter , while ne α1-ARs . Preincue α1-ARs .1-ARs are not a homogenous group and consist of three subtypes . While pes . The cell culture medium consisted of RPMI 1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, 10 mg/mL streptomycin, 100 U/mL penicillin and selection antibiotic for stably transfected cells. pcDNA3.1+ plasmids expressing human α1A-AR and α1B-AR were obtained from the Missouri S&T cDNA Resource Center (#AR0A1A0001 and #AR0A1B0000). Cell transfection was performed with Lipofectamine 2000 reagent  according to the manufacturer’s instructions. Stably transfected cells were selected by G418  and assayed for the functional activity of their receptors. To assess the activity of α1D-AR, we used ChemiSCREEN™ Ready-to-Assay™ α1D adrenergic family receptor frozen cells  and a Fluo-4 Direct™ calcium assay kit .The rat PC12 pheochromocytoma cell line was obtained from the DSMZ repository. The cells were cultured at 37 °C in a humidified incubator with 5% CO® assay kit . Cells with stable expressions of α1A- or α1B-AR were removed with a mix of PBS-Versene  (1:1), centrifuged for 5 min (200× g) and suspended in stimulation buffer . A total of 14,000 cells resuspended in 7 µL were seeded in each well of a 384-well plate. The next steps were performed according to the manufacturer’s protocol.The IP accumulation assay was performed with the IP-One HTRF(1)Noradrenaline or other agonist stimulation: 7 μL of consecutive concentrations of agonist in stimulation buffer was added to each well.(2)−10 to 4 × 10−4 M) of drug in stimulation buffer were added to each well, and after 5 min, 3.5 μL of noradrenaline was added for a final concentration of 4 μM (EC90).Antagonistic properties of drugs: 3.5 μL of various concentrations (from 4 × 1090 (antagonistic property measurement) response.After 75 min of incubation at 37 °C, the detection reagent was added, and after 2 h of incubation at room temperature, the time-resolved fluorescence at wavelengths of 620 nm and 655 nm was measured. IP One concentration was calculated according to the manufacturer’s instructions. The results are presented as percentages of the maximal (agonistic stimulation measurement) or ECChemiSCREEN™ Ready-to-Assay™ α1D adrenergic family receptor frozen cells  were cultured in DMEM  supplemented with 10% FBS. They were removed with trypsin/EDTA  and resuspended in assay buffer .A Fluo-4 Direct™ calcium assay kit  was used per the manufacturer’s instructions. Briefly, cells were plated in 5 µL of assay buffer at a density of 17,000 cells/well in standard tissue culture-grade, clear-bottom, black 384-well plates. The cells were incubated for 1 h at 37 °C, and the test compounds were added to the cells.(1)Stimulation with noradrenaline: 5 μL of consecutive concentrations of noradrenaline in stimulation buffer was added to each well.(2)−10 to 4 × 10−4 M) of drug in stimulation buffer were added to each well, and after 5 min, 2.5 μL of 1 μM NE (EC90) was added.Antagonistic properties of drugs: 2.5 μL of various concentrations (from 4 × 1090 (antagonistic property measurement) response.After 5 min, 10 µL of the 2X Fluo-4 Direct™ calcium reagent loading solution was loaded into the cells for 30 min at 37 °C and 1 h at room temperature. The fluorescence was measured with excitation at 494 nm and emission at 516 nm. The calcium ion concentration was calculated according to the manufacturer’s instructions. The results are presented as percentages of the maximal (agonistic stimulation measurement) or ECCells were seeded at 60% confluence (for 24 h incubation) or at 25% confluence (for 5-day incubation) in standard growth medium with 10 μM antidepressant or vehicle. After 24 h or 120 h, the cells were washed and harvested for IP1 or calcium ions measurement after stimulation with NA as described above.All experiments were performed in duplicate and repeated at least 4 times.g) at 4 °C. The supernatant was removed and homogenization and centrifugation steps were repeated. Obtained pellets were dissolved in buffer and frozen at −80 °C. Subsequent measurements were performed in 96 well-plates. For total binding, 25 µL of buffer , 25 µL of 8 [3H]-Prazosin (Perkin–Elmer) concentration (from 0.125 to 16 nM) and 150 µL of diluted pellet were used. For nonspecific binding, the buffer was replaced with 25 µL of 10 µM Prazosin (Sigma). After 1 h of incubation with shaking at 37 °C, the probes were harvested into 96-wells plates with GF/B filter (Perkin-Elmer) and washed 4 times. Subsequently, after overnight incubation in the dark, 35 µL of scintillate  was added, and luminescence was measured. The protein concentration in pellet form was diluted with the Lowry method. The protein concentrations were transformed into cell numbers from experimentally calculated formula, whereby 0.25 mg of protein comes from 1.1 million cells. Experiments were conducted 4 times in tetraplicates.The PC12 cells with stable expressions of α1A- or α1B-AR and ChemiSCREEN™ Ready-to-Assay™ α1D cells were washed and resuspended in 50 mM Tris-HCl, pH 7.4 buffer, followed by disruption in the tissue homogenizer Polytron and centrifugation for 45 min , citalopram hydrobromide (C7861), desipramine hydrochloride (D3900), mianserin (M2525) and imipramine hydrochloride (I7379) were purchased from Sigma-Aldrich . Fluoxetine hydrochloride (0927) and reboxetine mesylate (1982) were purchased from Tocris Bioscience .(1)The one-site specific binding curves were fitted to total nonspecific binding curves, and the Kd and Bmax parameters were calculated;(2)p < 0.05 was reached, in which case a 4-parameter model was chosen;The agonist dose–response curves were fitted with 3-parameter (log(agonist) vs. response) and 4-parameter (log(agonist) vs. response–variable slope) models. Model accuracy was compared with the F test, and a simpler (3-parameter) model was chosen unless a significance threshold of (3)p < 0.05 was reached, in which case a 4-parameter model was chosen.For fitting the inhibitory effects, the dose–response curves were fitted with 3-parameter (log(inhibitor) vs. normalized response) and 4-parameter (log(inhibitor) vs. normalized response-variable slope) models. Model accuracy was compared with the F test, and a simpler (3-parameter) model was chosen unless a significance threshold of The analysis was performed with GraphPad Prism 5.0 (binding assay) or GraphPad Prism 9.0 (the remaining experiments):50 values were calculated with the multiple t test with the Holm–Šidák correction. The logEC50 and maximal response values were compared with 2-way ANOVA with the Holm–Šidák multiple comparison test.Statistical differences between logICTo exclude the possible adverse effects of drugs on cells, metabolic activity was measured. The cells were seeded on 96-well plates at densities of 40,000 cells/well (for 30 min and 24 h incubations) or 20,000 cells/well per well (for 120 h incubation) for α1A- or α1B-AR-expressing cells, or at densities of 20,000 cells/well (for 30 min and 24 h incubations) or 10,000 cells/well (for 120 h incubation) for α1D-expressing cells. The cells were incubated with antidepressants at a 10 µM concentration for the indicated times. Metabolic activity was measured with a resazurin assay  according to the manufacturer’s protocol.


Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.NCT01942837  and NCT02025010 ) were used to further validate these findings.A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board–approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer . In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival , prostate-specific antigen progression-free survival , and radiographic progression-free survival .Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials. Conversely, some patients with CRPC develop AR-independent, neuroendocrine prostate cancer (NEPC) as an alternate escape pathway from androgen blockade.15 Comprehensive understanding of the molecular drivers of ARSI resistance and the ability to monitor this evolution over time is critical for early detection and appropriate treatment selection to improve outcomes for these patients.Androgen Receptor (AR) signaling inhibitors (ARSIs), including abiraterone acetate (AA), enzalutamide, apalutamide, and darolutamide, have improved survival for men with metastatic castration-sensitive prostate cancer (CSPC) and castration-resistant prostate cancer (CRPC).Key ObjectiveMultiple mechanisms of resistance result in disease progression in men with prostate cancer treated with androgen receptor (AR) targeted therapies . This study aimed to develop a multiplex liquid biopsy that could detect resistance from AR-driven and AR-independent mechanisms to identify diverse drivers of treatment resistance.Knowledge GeneratedA pattern of gene expression was identified by hierarchical cluster analysis from RNA extracted from circulating tumor cells (CTCs) from patients with prostate cancer. This cluster was shown to be prognostic for decreased overall survival in the training cohort and decreased progression-free survival and overall survival in the validation cohort including patients from two clinical trials with ARSI.RelevanceDetection of androgen receptor splice variant 7 in CTCs has emerged as a biomarker that is prognostic for treatment resistance to ARSI. Here, we demonstrate that expression of AR-regulated genes in CTCs is also prognostic for survival and radiographic progression that may have broader ability to identify treatment resistance than androgen receptor splice variant 7 alone.19AR-V7 expression or protein in CTCs can be predictive of response to ARSIs such as enzalutamide and abiraterone acetate (AA).26 In the PROPHECY trial, which profiled 118 patients with CRPC, 0% and 11% of patients who had detectable CTC AR-V7 by the Epic Sciences nuclear AR-V7 protein assay or the Johns Hopkins AR-V7 mRNA assay, respectively, had confirmed prostate-specific antigen (PSA) responses. The AR-V7–positive patients also had significantly worse progression-free and overall survival (OS) as compared with patients who lacked detectable AR-V7. The prevalence of AR-V7 is low in the first-line setting and many AR-V7–negative patients do not respond to second-line ARSIs, suggesting other mechanisms of resistance.28 NEPC is characterized by the loss of AR transcriptional activity in tandem with upregulation of neuroendocrine markers and is observed in 5%-17% of men with CRPC.29 NEPC has been previously identified using ctDNA methylation profiling and morphologic characterization of CTCs.31 To our knowledge, there is no assay capable of assessing AR-Vs, AR activity, and NEPC on a single platform, which is required to understand the timing and emergence of these diverse resistance mechanisms that occur and interact.Molecular profiling to detect these resistance mechanisms has historically required serial tumor biopsies, which carry a risk of procedural complication and high likelihood of not obtaining adequate tissue for analysis. The predominance of bone metastases in prostate cancer and the process of decalcification for pathologic assessment also render it challenging to assess RNA expression in metastatic bone biopsy samples. Liquid biopsies, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), have shown prognostic relevance in CRPC and the potential to be used as a surrogate biomarker of survival.AR-V7 and AR-V9), a panel of AR-regulated genes  that may indicate persistent or overactivation of AR transcriptional activity, and NEPC-associated genes (SYP and CHGA). We applied this assay to a large multi-institutional cohort of patients with metastatic prostate cancer where we observed associations with clinical outcomes and described longitudinal changes compared with changes in ctDNA. These findings were subsequently independently validated in two multicenter phase II trials.Herein, we present a CTC liquid biopsy transcriptional assay capable of simultaneously measuring these three resistance mechanisms. This multiplex panel includes AR splice variants . Patients were required to have a histologically confirmed metastatic prostate cancer diagnosis. All patients gave informed consent following an institutional review board–approved protocol to participate in prospective blood sample collection and analysis. Study data were managed using approved REDCap electronic database hosted at the University of Wisconsin (UW)–Madison, School of Medicine and Public Health.NCT01942837) was a phase II trial of enzalutamide in metastatic CRPC (mCRPC), which accrued 66 patients from 2013 to 2017. The AA-CRPC trial  was a phase II trial of AA without glucocorticoid therapy for men with mCRPC,33 which accrued 58 patients between 2014 and 2016. Liquid biopsies were prospectively collected before treatment and evaluable on 21 patients from ENZA-CRPC and 27 patients from AA-CRPC, for a total of 48 patients from prospective phase II trials treated with ARSIs. These trials served as independent validation for the C2 classifier.The ENZA-CRPC trial . Expression values of CTCs isolated from 15 mL of blood were calculated as 38 − Ct values and were reported as raw  data.Fifteen milliliters of blood was collected from each patient in EDTA vacutainer tubes . Samples were shipped in temperature-controlled packing and processed within 24 hours. The VERSA platform, as described previously,g for 10 minutes, spun at 2,730 × g for 10 minutes, and stored at –80°C. Cell-free DNA was isolated using the Qiagen Circulating Nucleic Acids kit following manufacturer's instructions. Cell-free DNA sequencing was performed as previously described.36Plasma was collected from whole blood collected in EDTA tubes within 4 hours of draw and before isolation of CTCs. Plasma (6 mL) was removed after centrifugation at 290 × 37 was trained using the same genes on the baseline CTC samples using the R pamr package with default parameters. This approach computes a centroid for each cluster, applies a shrinkage step to reduce noise, and classifies new samples on the basis of the nearest centroid. This was then locked and used to classify the ENZA-CRPC and AA-CRPC samples . For comparisons between two clusters, Fisher's exact test and Mann-Whitney U test were used for categorical and continuous variables, respectively. OS was the primary clinical end point and was defined as date of death or last contact relative to treatment start. PSA progression-free survival (PSA PFS) was defined as date of PSA progression according to the Prostate Cancer Working Group 2 criteria or death because of any cause or censored at date of last disease assessment relative to treatment start. Radiographic PFS (rPFS) was defined as radiographic progression according to RECIST 1.1 criteria for soft tissue and lymph node disease and Prostate Cancer Working Group 2 for bone disease or death because of any cause or censored at date of last disease assessment relative to treatment start. The Kaplan-Meier method was used to estimate the survival distributions by clusters and AR-V status, and log-rank test was used to compare two groups. Univariate and multivariate Cox proportional hazards models were fitted to quantify the association of molecular and clinical variables with time-to-event end points.Hierarchical clustering of the gene expression data from the UW-DFCI samples (training set) was performed using the R hclust function with default parameters to identify distinct clusters. To create a single-sample approach to independently classify samples in the phase II trials, a nearest shrunken centroids classifierBlood samples from 99 unique patients with metastatic prostate cancer were included in the initial cohort. Patient characteristics and treatment history at the time of blood sample collection are detailed in Table AR, AR-Vs, AR-regulated genes, and genes upregulated in NEPC using RNA isolated from CTCs captured with an anti-EpCAM antibody. Hierarchical clustering of the transcript expression from these samples identified two distinct clusters of patients  . As has been reported,26 AR-V+ patients (AR-V7 and AR-V9) had worse OS . On multivariable analysis (MVA), C2 was independently associated with shorter OS after adjusting for patient age, CRPC versus CSPC, NEPC versus CSPC, PSA, visceral metastasis, and AR-V status. CRPC and NEPC were the only other variables also independently associated with OS on MVA , suggesting that AR-V status may be prognostic because of its association with C2, rather than being independently prognostic. When we examined only C2 patients, no significant differences in OS were detected on the basis of AR-V status (Data Supplement). However, when we examined only AR-V+ patients, significantly shorter OS was observed for C2 patients compared with C1 patients (Data Supplement). This result is consistent with the MVA and suggests that these transcriptional clusters are inclusive of AR-Vs, and the prognostic importance of AR-Vs alone is eliminated when taking into account CTC gene expression clusters.We next examined the clinical end point of OS. At time of analysis, 55 patients (56%) were deceased and the median follow-up time was 9.4 months in this cohort. We observed significantly shorter OS for patients in C2 versus C1     treated with ARSIs, enzalutamide (ENZA-CRPC), or AA-CRPC. At time of analysis, 15 patients (31%) were deceased and the median follow-up time was 28 months in this cohort. We applied the classifier trained in UW-DFCI to these cohorts to independently identify and clinically validate C2-like patterns in CTCs. In the combined trials of ENZA-CRPC and AA-CRPC N = 48), we identified 10 patients (21%) with a C2 expression pattern. This C2 cluster was associated with worse OS ] months 8, we ideSYP, MYCN, and CHGA 3 months before clinical symptoms ,50 or even bypassing AR completely via glucocorticoid receptor.51 C2 appears to be primarily driven by increased expression of AR-regulated genes, whereas total AR levels do not differ dramatically. Interestingly, our work suggests that prior interest in AR-Vs may be incomplete. AR-V positivity was correlated with C2, but after adjusting for C2 membership, AR-V status was no longer prognostic. This may be because multiple mechanisms can result in ARSI resistance and manifest in increased AR signaling activity, including not only AR-Vs but also from a myriad of other etiologies. We also independently validated that C2 was associated with worse prognosis in two phase II ARSI trials. Strikingly, C2 was also associated with worse PSA and rPFS with ARSIs, suggesting that it could be used as a biomarker to identify patients who may benefit from additional or alternative therapies up-front and monitor for the early emergence of molecular resistance. These findings warrant further exploration in larger prospective trials.Many diverse mechanisms of resistance to androgen blockade culminate in increased expression of AR target gene expression. These include mutations, amplifications, and rearrangements of the AR gene;18 The PROPHECY trial demonstrated that AR-V7 expression, either by gene expression and protein analysis, is prognostic for PFS and OS with ARSI treatment independent of enumeration.26 It is unknown if the number of CTCs present in each sample may affect the detection or level of gene expression for the genes in this panel. Further analysis is necessary for the panel in this report to evaluate the relative contribution of CTC number to this assay. In addition, broader evaluation of non–AR-driven resistance mechanisms, such as the recently described Double Negative Prostate Cancer classifier, may identify a distinct population of patients with different outcomes following treatment with ARSIs. Expansion of this gene expression panel to incorporate newly identified disease subtypes and mechanisms of resistance may further enhance the utility of liquid biopsies.Previous studies of CTCs in CRPC identified other prognostic signatures that associate with clinical outcomes. CTC enumeration has been shown to be prognostic for OS in prostate cancer using Cell Search platform.Molecular classifiers such as the approach described in this study also represent an attractive way to follow prostate cancer disease status in addition to PSA and imaging. These could be easily integrated into existing clinical workflows, such that, in addition to standard periodic laboratory studies, blood samples could be sent for real-time liquid biomarker studies. We demonstrate the feasibility and concordance of longitudinal CTC and ctDNA profiling simultaneously. Identification of molecular mechanisms of ARSI resistance could trigger a change in therapy or enrollment into clinical trials. Early detection maximizes eligibility for other treatments, and although tumor burden is still low , the patient's performance status is still optimal, before symptomatic disease progression.NCT02445976, NCT01942837, NCT03725761, NCT02025010, and NCT04126070).In conclusion, to our knowledge, we demonstrate the first multiplex gene expression liquid biopsy assay that can assess multiple potential ARSI resistance mechanisms simultaneously in metastatic prostate cancer. Early identification of these molecular changes could help guide treatment decisions. The heterogeneous nature of this cohort, which included different disease states and therapies, suggests that these findings may be generalizable to the broader patient population. The validation of our prognostic clustering in two phase II trials with ARSIs suggests clinical utility; however, larger clinical trials are necessary. We are prospectively testing these findings further in multiple ongoing clinical trials for patients with CSPC and CRPC (


The COVID-19 pandemic has demonstrated how a stochastic disruptive event can dramaticallyalter community soundscapes. Whilst religious bells have symbolism in many worldwidefaiths, the sound emanating from church bells can be considered public domain andtherefore, is not exclusive to the church. Pandemic-related interruption of these soundsimpacts not only the church involved, but both the surrounding soundscape and any membersof the community who ascribe value to these sounds. This paper examines the soundscape ofChristian churches in the states of New South Wales and Victoria, to give an Australianperspective one year after the declaration of the COVID-19 pandemic in March 2020. Itprovides an update of the situation in Australia, building on our previous work fromAugust of that year. In doing so, it explores the activity of church tower bell ringing,and how this “non-essential” activity has been affected, both during and subsequent to theheavy community restrictions applied in Australia. The paper also explores what lengthsbellringers have undertaken to be permitted to conduct such activities, such as the use ofadaptive measures due to “social distancing”, and considers what implications thisenforced silence has in similar soundscapes elsewhere in the world. I.In 2018–2019, the authors undertook a study examining church bell ringing in the state ofNew South Wales, Australia, with research questions investigating the extent of church bellringing still practiced, what factors may determine this differentiation, and what valuesand significances were attributed to the bell ringing sounds by the practitionersthemselves. While the full data is reported elsewhere , we founIn August 2020, the authors investigated the effects of COVID-19 on a sub-set of theinitial cohort, with specific interests in how bell ringing sounds had changed over sixmonths in NSW . ThistiII.While the previous study looked at both single bells (tolling bells) and tower bells, wedecided to follow up with a study in 2021 solely pertaining to tower bells, due to readilyavailable data sources. The Australian and New Zealand Association of Bellringers (ANZAB) isa long-running (50 y) community entity promoting the art of change ringing, which maintainsa complete register of towers containing ringing bells in these two countries. With theonset of the COVID-19 pandemic, they promptly set up a section of their website discussingwhich towers would be open and which towers would be closed due to differinggovernmental-imposed restrictions in the listed towers. To confirm data quality, individualcontact with tower captains or other relative personnel was then made in cases of suspectederrors on the website, as was the use of other public data, such as web pages and socialmedia accounts. Data were collected on average bi-monthly, with follow up and verificationvia direct communication at the time of manuscript development. This information produced anaccurate record showing when each individual tower opened up, over the period March 2020 toApril 2021. We limited our study to the two most populous states in Australia—New SouthWales and Victoria—with NSW having 16 towers in the capital city of Sydney and another 16 inregional towns, and Victoria having five towers in the capital city of Melbourne and anotherfive towers in regional areas of this state.III.Over the entire annual period from late March 2020 to April 2021, we found that tower bellringing in both NSW and Victorian churches was highly correlated with governmental-imposedrestrictions which stipulated actions allowable by the community. Previously to any COVIDimpacts or restrictions, all of the bell towers in these two states listed by ANZAB wereopen and ringing as per normal. The results for NSW and Victoria  had different patterns, which can be attributed to the different restrictionsimposed in either state.2, and finally, to one person per2 m2 in April 2021. There was a much larger increase in church bell towerringing over this time in the churches of Sydney, with levels of bell ringing approaching100% (when these new restrictions were implemented) and hitting the full capacity ofpre-COVID levels by the week of April 14, 2021. Regional churches showed a similar patternof decline and return to bell ringing, although the lag times were greater than inmetropolitan areas: the return to 30% capacity did not eventuate until August, and the finalfull capacity did not eventuate until April 21, 2021  and five towers in regional areas, compared to the 16 each in NSW. The Victorianshutdown began largely in the same way to NSW, with the first community transmission onMarch 7, followed by an initial gradual lockdown, and then a snap nationwide lockdown forsix weeks . Similar2 capacity rule inJanuary 2021. There was an immediate positive response in bell ringing in Melbourne with areturn to 60% of pre-COVID levels. In early February 2021, a case of community transmissioncaused a one-week snap lockdown across the state, and unlike the previous lockdowns of agradual reduction of restrictions, this lockdown was sharp enough to allow a rapid bounceback to pre-levels, both in capacity limits and the prevalence of tower bell ringing. Fullreturn of bell ringing to pre-COVID levels occurred by early April 2021 in Melbourne.Churches in regional Victoria showed a similar lag pattern, with only 20% (one of fivebells) returning to ringing activities in the Christmas 2020 period, and only to 80% by theEaster period 2021  apart  apart . In a stt) apart . Suchclt) apart . Other cDuring the return to regular bell ringing, we discovered some really interesting adaptiveresponses being employed in some churches, both for bell training and in public bellringing. In order to continue some form of training for the bellringing team, some towergroups advocated for the use of “virtual ringing rooms” during the height of the pandemic,using web-based applications where the actual ringing action is undertaken by pressing a keyto ring an individual bell. It was noted that while it kept mathematical functions workingcognitively, it offered none of the actual physicality of ringing . One chuOther churches applied adaptive measures when actually ringing tower bells, in an attemptto return to the activity level pre-COVID, while still complying with restrictions. Forexample, at St Paul's Anglican Church, Burwood (NSW), eight bells were rung in the periodpre-COVID, which was the total maximum, as it is an eight-bell tower Fig. . During This presents an interesting case for discussion: the ringing of tower bells during theCOVID-19 pandemic is therefore not so much a question of the presence or absence of bells,but more a question of the richness of the sound that is able to be rung due to the numberof available bells as a result of space restrictions. The reason follows thus: Ringing acertain number of bells allows specific repertoire to be performed; eight bells allows theperformance of Basingstoke Surprise Major, and six bells allow Grandsire Doubles. However,limiting the number of bells actually restricts the repertoire available to be performed,and instead of scalic patterns, the church has to offer other suggestions, such as triadicpatterns or similar. These restrictions, therefore, do not only create limitations on thenumber of churches ringing bells, but a change in the manner of richness in the soundscapeof the surrounding area. Further adaptive measures allowed some churches to conquer this,such as the ringing of non-adjacent bells—there were cases of churches ringing Basingstokewith four bellringers, with bellringers having two bells each; and other towers utilizedfamily groups for ringing purposes as the restrictions in this situation were not as strict.We discovered similar instances of adaption. For example, Plain Bob was documented beingperformed having only three bellringers using two bells each, and Oxford Treble Bob Minorusing only three bells with three ringers, rather than the usual six bells.Furthermore, our research uncovered other aspects of change pertaining to the soundscapescreated by church bell ringing, including differences in duration and intensity of emanatedsound. Following government health advice recommending limiting the time spent within anindoor space, bell ringing duration was reduced from 1.5–1 h at weekly practice sessions atAll Saints Church, Singleton (NSW), and limited to 15 min solely for services at HoskinsUniting Church, Lithgow (NSW) under a specially devised COVID-19 Safety Plan . Of partAt the time of writing (September 2021), it is important to note that parts of Australiahave again returned to a total shutdown of non-essential activities due to increased casesof community transmission of the COVID-19 virus. Greater Sydney has been shut down for atleast eleven weeks and regional NSW for three weeks, Melbourne for five weeks, and regionalVictoria for two weeks. It is expected that an enforced lockdown will continue for some timeinto the future, and with it, a continued hiatus of tower bell ringing across bothmetropolitan and regional areas of NSW and Victoria. However, it is important to note thatthe cessation of church bell ringing is in no way limited to states of Australia. As ofApril 2021, one year from the onset of COVID-19, it was reported that there had only been atotal of five tower bell peals rung throughout the world, and all of these had been rung inNSW , and of the 99 tower bell quarter pealsrung worldwide, 68 had been rung in towers associated with the Australian and New ZealandAssociation of Bellringers (ANZAB) .Indeed,V.Clearly, COVID-19 had quite a dramatic impact on the practice of bellringers in both NSWand Victoria and the way in which they could practice their art. It basically silenced thetowers for the entire initial lockdown period and then subsequent periods. Even if towersreopened due to capacity restriction changes, restrictions limited the number of peoplebeing allowed to engage and participate in the bell ringing activity, so any regularpracticing activity had been effectively ceased. Whilst gradual return allowed therecommencement of the practicing activity, we must keep in mind that as the art form of bellringing requires excellent timing, missing almost one year's worth of physical practicecould negatively impact any art community. We show in this example that COVID-19 had apotentially massive impact on community wide soundscapes, first in the silencing of bellsounds; and in cases where sounds were effectively permitted, such as the ringing of anindividual tolling bell, the rich sound created by tower bells was basically silenced. Whilewe know these sounds did gradually return, at the moment we do not know what the impact ofthis change in soundscape had on the community well-being. Whilst we can report and discussstatistics whether churches did or did not ring, social science studies have not instigatedresearch looking at the effect of lack of sound on people. We also need to recognize that wecurrently do not have any pre-COVID measurements of community well-being scales either. Thispresents opportunity for future research, and these are activities that we shouldinvestigate in the current climate, as it would be prudent to assume that the COVID-19pandemic will not be the last pandemic to affect our society.


Ovarian cancer (OVCA) arises from three cellular origins, namely surface epithelial cells, germ cells, and stromal cells. More than 85% of OVCAs are EOCs , which are the most lethal gynecological malignancies. Cancer stem/progenitor cells (CSPCs) are considered to be cancer promoters due to their capacity for unlimited self-renewal and drug resistance. Androgen receptor (AR) belongs to the nuclear receptor superfamily and can be activated through binding to its ligand androgens. Studies have reported an association between AR expression and EOC carcinogenesis, and AR is suggested to be involved in proliferation, migration/invasion, and stemness. In addition, alternative AR activating signals, including both ligand-dependent and ligand-independent, are involved in OVCA progression. Although some clinical trials have previously been conducted to evaluate the effects of anti-androgens in EOC, no significant results have been reported. In contrast, experimental studies evaluating the effects of anti-androgen or anti-AR reagents in AR-expressing EOC models have demonstrated positive results for suppressing disease progression. Since AR is involved in complex signaling pathways and may be expressed at various levels in OVCA, the aim of this article was to provide an overview of current studies and perspectives regarding the relevance of androgen/AR roles in OVCA. BRCA1 and BRCA2, and age; estrogenic influences, such as early menarche and menopause after 52 years of age; environmental risk factors; and lifestyle choices, such as exposure to polycyclic aromatic hydrocarbons (PAH), cigarette smoking, and obesity [Ovarian cancer (OVCA) is among the most lethal gynecological malignancies and has a variety of cellular origins, histological characteristics, and therapeutic responses . Despite obesity .Androgen receptor (AR) was well-characterized as ligand-dependent transcription factor, which belongs to the nuclear receptor superfamily . Androge2+ concentration and cyclic adenosine monophosphate (cAMP) levels, respectively [AR has also been reported to become activated and translocate into the nucleus without binding to a ligand, which is referred to as “non-classical transactivation” or “ligand-independent transactivation” ,12,13,14ectively ,16,17.In cancer biological research, AR has been reported to be expressed in many cell types, and androgen/AR signaling has been found to promote tumorigenesis and metastasis in several cancer types, including OCVA ,19. AccuSex-cord/stromal tumors develop from the stroma or the sex cord. The stroma and sex cord are tissues that support the ovary and produce the female hormones estrogen and progesterone. Stromal tumors are a rare tumor type, comprising less than 7% of all OVCAs. Sex-cord/stromal tumors can be identified at any age but most commonly affect young women (median age 27 years), causing abnormal vaginal bleeding and pain. Surgery is the primary treatment option for stromal tumors, and the prognosis is generally favorable ,24. AlthOvarian teratoma (OVTC) arises from germ cells and is typically benign. Malignant teratoma, known as teratocarcinoma, is a minority tumor type (less than 10%) of the OVTC . OVTC map53 mutation-associated, with high p16 expression and the loss of BRCA1 expression. HGSC is often initially chemosensitive, followed by the development of resistance and poor prognosis, whereas low-grade SC (LGSC) is associated with a noninvasive, serum-confined, less proliferative, and KRAS and BRAF mutation-associated, with generally intermediate chemosensitivity, except metastasis [KRAS mutation (43.6%) and HER2 overexpression (18.8%) [CTNNB (β-catenin), PI3CA (PI3K), and PTEN (phosphatase and tensin homolog) have also been reported to be EC risks. Similar to LGSC, EC is highly sensitive to chemotherapeutic agents and associated with better prognosis in patients [ARID1A genes and upregulation of HNF-1 protein [More than 85% of OVCA are EOCs, which are the most lethal gynecological malignancies that arise from ovarian surface epithelial cells (OSECs) . The foutastasis ,39,40. Itastasis ,38. MC h 18.8%) ,42. It h8.8% ,42.mutation .6% and Hpatients ,38. The patients ,38. GeneAlthough EOC represents a heterogeneous disease, decisions regarding standard therapeutic strategies continue to rely on the diagnosed stage and grade rather than histological type . The curSome factors had been associated with poor prognosis of EOC, including (1) complicated personal risk factors, such as individual diet, inherited gene mutations, and ages; (2) a lack of early diagnosis markers; and (3) resistance to chemotherapy. Thus far, several studies have focused on the relationships between EOC treatments and these factors, both in vivo and in vitro.2+ concentrations and cAMP levels [The AR protein structure consists of three major functional domains: N-terminal domain (NTD), DNA-binding domain, and ligand-binding domain. The NTD exerts a modulatory function during protein–protein interactions, such as those with coactivators or other transcription factors . The DNAP levels ,16,17. IP levels ,16, and P = 0.03) were significantly higher in the cancer patients than in the control subjects [P = 0.01), specifically SC , in postmenopausal women [P = 0.02) and androstenedione , was observed in premenopausal women (n = 13), but not in postmenopausal women (n = 18).) [EOC has been shown to be an androgen-responsive tissue ,53. Andrsubjects . The bloal women ,57 TheseStudies showed that AR expression varied across histological EOC subtypes. Recent studies showed that AR is expressed in all EOC subtypes, but higher expression levels were observed in SCs than in other EOCs ,22. ThisAR (hAR) mutations in 38 human OVCA cell lines associated with different AR expression patterns, and the results showed no mutations in the hAR gene. They concluded that mutation screening of hAR might not provide any information for OVCA risk assessment [AR is the conventional cellular receiver for androgens. The AR gene is composed of eight exons. Exon 1 contains two polymorphic trinucleotide repeats: a 9–39 CAG repeat  and a 14–27 GGN repeat  ,61. Manysessment .Accumulated studies have indicated that androgen or AR activity can affect OVCA progression and could serve as potential therapeutic targets for the treatment of this disease. Syed et al. (2001) found that testosterone and DHT significantly stimulated cell growth in both malignant and normal ovarian cell lines . This anAndrogen-induced EOC proliferation may be partially mediated by enhanced interleukin (IL)-6 and IL-8 expression, which might promote EOC growth via the activation of the AR gene promoter . Ligr etCSPCs are thought to be responsible for cancer phenotypes, including pathogenesis, metabolism, metastasis, drug resistance, and relapse . Previou+ cells) facilitated OVTC cell growth [Numerous studies have demonstrated that AR regulates progression of cancer stem cell in various cancer types, including OVCA . A studyl growth . Chen etl growth . Lin et l growth . These sChemoresistance remains a major challenge to cancer chemotherapy. Most patients respond to chemotherapies during the initial treatment period combined with surgery; however, many patients exhibit a limited response to chemoreagents and become resistant to subsequent treatments ,50. Acco+ OVCA [In addition to the role played by AR in OVTC CSPCs, Chung et al. (2019) also showed that AR regulated the expression of ABCG2, an MDR-associated membrane protein, in SC cells under paclitaxel treatment conditions through ligand-independent AR translocation activity. Furthermore, treatment with ASC-J9, an AR degradation enhancer, eliminated the paclitaxel–AR–ABCG2 axis and enhanced drug sensitivity in OVCA cell lines. Chung et al. showed that targeting the degradation of AR protein is more beneficial than anti-androgen to treating AR+ OVCA .AR has been reported to be differentially expressed across EOC subtypes ,22, and The early clinical reports examining the effects of flutamide, bicalutamide, and goserelin have been conducted in OVCA patients. The phase II clinical trial examining the use of flutamide in 68 EOC patients treated with platinum-based chemotherapy was reported by Tumolo et al. (1994) . In thisMore recently, a phase II study of bicalutamide  and goserelin (3.6 mg subcutaneously every 4 weeks) in stages III and IV EOC patients was reported by Levine et al. (2007) . A totalAccording to these clinical trials, only a few EOC patients were responsive to anti-androgen therapy combined with other chemotherapies. However, the results of anti-androgens  that block the androgen/AR signaling axis in clinical trials have been controversial, and these treatments have been ineffective for curing most patients of this difficult-to-treat disease . One posAn AR degradation enhancer, dimethylcurcumin (ASC-J9), has also demonstrated effective tumor inhibition, including against OVCA. Lin et al. (2018) showed that ASC-J9 inhibitor promoted EOC cell proliferation, migration, and sphere formation in CSPCs in vitro and in vivo . Thus faBRCA1 or BRCA2 mutations, approximately 50% of patients are diagnosed with tumors that exhibit homologous recombination (HR) deficiencies due to either a germline or somatic mutation in the HR system [BRCA1/2-mutated advanced OVCA who had received three or more chemotherapies had a median PFS of 7 months, a median overall survival (OS) of 16.6 months, and a 1-year OS rate of 64.4% [P < 0.001) [Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) represent the first FDA-approved treatment to apply the concept of synthetic lethality (cell death induced by single strain break) to the treatment of EOC. Although only 10–15% of patients with high-grade serous OVCA (HGSOC) harbor germline R system . The resof 64.4% . Studies< 0.001) ,104.BRCA1 [Several studies have reported that treatment with the PARPi olaparib, combined with enzalutamide and olaparib, demonstrated synergistic effects in prostate cells and orthotopic xenograft models ,106. In BRCA1 . In our BRCA1 . Thus, i+ OVCA subtype. Discerning the intricacies and crosstalk between AR and the tumor microenvironment may also provide advantages for OVCA treatment and would not only advance our understanding of the role played by AR in cancer progression but also identify new treatment strategies through which AR signaling can be blocked to improve outcomes for women with OVCA. A new perspective on combination therapy of AR degradation and chemoreagents is illustrated in Although the role played by AR in OVCA has been studied in vitro, providing additional understanding regarding this relationship, the development of an effective in vivo model for AR research and combinational therapy for use in preclinical trials for this disease remains an active and increasingly popular area of investigation. To better understand the mechanism of AR signaling and to design proper therapies against AR in OVCA, an increased focus must be placed on identifying the AR activation mechanisms and target genes that contribute to tumor recurrence, and the development of therapy resistance is necessary. Advancements in this mechanistic understanding will shed light on potentially effective combination therapies for patients with the AR


Background: Comprehensive rehabilitation programs are recommended for patients with prolonged mechanical ventilation (PMV) to facilitate functional recovery and ventilator weaning, but whether the functional status after rehabilitation influences outcome has not been clearly evaluated. This study aimed to investigate the association between post-rehabilitation functional status and weaning and survival outcome in PMV patients.Methods: We retrospectively enrolled PMV patients admitted to the respiratory care center (RCC), a post-ICU weaning facility with protocolized rehabilitation program, from January 2016 through December 2017. Functional status was measured by the de Morton Mobility Index (DEMMI), with a cut-off value set at 20 points. The primary outcomes were the weaning status at RCC discharge and hospital survival. The secondary outcomes were overall survival and survival at 3 months after RCC discharge. We followed patients until 3 months after RCC discharge or death. Logistic and Cox regressions were performed to identify significant parameters associated with weaning success and survival.Results: In total, 320 patients were enrolled. The weaning success rate was 71.6%. The survival rate at RCC discharge, hospital discharge, and 3 months after RCC discharge was 89.1, 77.5, and 66.6%, respectively. Post-rehabilitation DEMMI ≥ 20  was the most significantly associated with weaning success. The weaning success and higher post-rehabilitation DEMMI were the two most significant independent factors associated with both hospital survival  and survival at 3 months after RCC discharge . Post-rehabilitation DEMMI ≥ 20 remained significantly association with overall survival at 3 months after RCC discharge .Conclusions: Post-rehabilitation functional status of PMV patients was independently associated with weaning success, as well as hospital and 3-month overall survival after RCC discharge. Post-rehabilitation, but not pre-rehabilitation, functional status was a significant parameter associated with weaning success and survival in patients requiring PMV. One of the leading causes for intensive care unit (ICU) admission is acute respiratory failure with mechanical ventilator use . Due to Pulmonary rehabilitation with early mobilization and physiotherapy has been proposed as a component of multidisciplinary strategy to promote successful weaning in patients with PMV use . SeveralThe aim of this study is to evaluate the association of functional status after pulmonary rehabilitation in PMV patients and the weaning and survival outcome. We hypothesize that functional status after pulmonary rehabilitation is associated better weaning and survival rates.2O, and (4) stable hemodynamic conditions, with norepinephrine-equivalent dose ≤ 5 μg/min. Patients who survived but failed ventilator weaning after a 6-week stay in the RCC were transferred to long-term care facilities. The nurse-to-patient ratio in the RCC was 1:4, and one respiratory therapist is on duty during each shift. All patients transferred to the RCC received standardized respiratory therapist-driven protocol for ventilator weaning (This study was conducted retrospectively in the 12-bed respiratory care center (RCC), a step-down and protocol-driven weaning facility, in the National Taiwan University Hospital Hsin-Chu branch, a university-affiliated hospital in northern Taiwan. PMV was defined as ventilator support for more than 21 days, according to the definition from the National Association for Medical Direction of Respiratory Care Consensus Conference in 2005 , and the weaning . All eliAll patients received the same physical training program conducted by experienced physical therapists 5 sessions per week during the entire RCC stay. The standardized session of physical training program included: (1) breathing exercise  in semi-recumbent position (≥45°), followed by cough training, with 10 repetitions per set for 1–2 sets; (2) chest mobility exercise, containing manual technique, and arm elevation with upper trunk lateral flexion, with 10 repetitions; (3) strengthening exercise for respiratory, abdominal and upper and lower limb muscles, containing antigravity elevation and resistance band exercise (for upper limbs), or straight leg raising or bridging exercise (for lower limbs), with 10 repetitions per set for 2–3 sets; (4) range-of-motion exercises  of upper and lower extremities, with 10 repetitions; (5) functional activity training , with 1–2 repetitions; and (6) endurance training, containing sitting/standing for 10 min, or limbs exercise/biking for 5 min. Each session lasted for 15–50 min (as long as tolerated), with all 6 elements performed as much as possible, according to individual tolerance and program progression. In neurologically impaired patients that could not follow orders, the endurance training was not performed.Functional status was measured by the de Morton Mobility Index (DEMMI), originally developed to assess the mobility status in hospitalized elderly patients . It was DEMMI cut-off level for analysis in our study was determined according to the following steps and rationale. First, the corresponding optimal cut-off values determined by Youden index point on the receiver operating characteristic (ROC) curves of post-rehabilitation DEMMI were 17.5 , 11.5 , and 11.5  for weaning success, hospital survival, and 3-month survival after RCC discharge, respectively. Also, the reported minimal clinically important difference for DEMMI ranged from 10 to 20 points –16. The Imax), maximal expiratory pressure (PEmax), rapid shallow breathing index (RSBI), tidal volume, and minute ventilation, were measured twice, at RCC admission (pre-rehabilitation) and before extubation or RCC discharge (post-rehabilitation), respectively. The established cut-off values for weaning evaluation were used as references, including PImax: −20 cm H2O, PEmax: +30 cm H2O, RSBI: 105, tidal volume: 5 mL/kg, and minute ventilation, 10 L/min  at RCC discharge and survival status at hospital discharge. The secondary outcome measurements were survival at 3 months after RCC discharge and overall survival after respiratory failure. Weaning success was defined as liberation from the ventilator (both invasive and non-invasive) for more than 5 days, according to the prospective payment system of ventilator dependents' managed care by the National Health Insurance in Taiwan . Patientt-test, and categorical variables were compared using Pearson's χ2 or Fisher's exact test, as appropriate. Multivariate logistic regression models were used to identify significant clinical factors associated with weaning success, hospital survival, and 3-month survival outcome in the study population. Kaplan–Meier curves were plotted for overall survival after respiratory failure, and the differences between patient subgroups were compared using the log-rank test. Multivariate Cox proportional-hazards model with adjustment for variables with statistical significance in the univariate analysis to identify significant clinical factors associated with overall survival after respiratory failure. Variables included in the logistic and Cox multivariate analysis were statistically significant factors in the univariate analysis. Both logistic and Cox regression analyses were performed with backward selection using the entry and stay criteria at 0.05 and 0.1, respectively. We used stepwise regression in order to manage large amount of potential predictor variables and to choose the best predictor variables from the available options. A two-sided P-value < 0.05 was considered statistically significant. All analyses were performed using SPSS version 18.0 for Windows .Data are expressed as the mean ± standard deviation and number (%) for continuous and categorical variables, respectively. Continuous variables were compared using Student's Between January 2016 and December 2017, 2,653 patients were admitted to our ICUs with mechanical ventilation support. Among them, 320 patients requiring PMV support were subsequently transferred to the RCC. The mean age was 70.9 years and 202 (63.1%) were men. Pulmonary disease (40.3%) and post-operative care (25.9%) were the two most common causes of respiratory failure. The baseline characteristics and demographic features of the study population were shown in P = 0.032). Similarly, while all the pre-rehabilitation weaning parameters did not differ between these 2 groups, patients with weaning success had higher post-rehabilitation PEmax  and lower post-rehabilitation RSBI  than those with weaning failure. The duration of rehabilitation did not significantly differ between patients with weaning success and those with weaning failure , or between groups with post-rehabilitation DEMMI ≥ 20 and those with post-rehabilitation DEMMI < 20 . Multivariate logistic regression models showed that the absence of chronic obstructive pulmonary disease , post-operative respiratory failure , post-rehabilitation DEMMI ≥ 20 , post-rehabilitation RSBI ≤ 105 , and higher platelet counts  were significantly associated with weaning success . Similarly, while all the pre-rehabilitation weaning parameters did not differ between survivors and non-survivors at hospital discharge or 3 months after RCC discharge, both of those survivors had higher post-rehabilitation PEmax than those non-survivors. The duration of rehabilitation did not significantly differ between patients with hospital survival and those with hospital mortality , or between groups with survival and mortality at 3 month after RCC discharge . Multivariate logistic regression models showed that weaning success, post-rehabilitation DEMMI ≥ 20, and platelets were significantly associated with both hospital survival  and 3-month survival after RCC discharge   .P < 0.001). The 3-month survival rate after RCC discharge surpassed 90 and 95% in patients with post-rehabilitation DEMMI ≥ 20 and ≥ 30, respectively  and survival  rate at various time points than those who still had post-rehabilitation DEMMI < 20 (rehabilitation non-responders).We also compared different weaning and survival status among patient groups stratified by different pre- and post-rehabilitation DEMMI combination, as shown in P < 0.001).Among patients with weaning failure, patients with post-rehabilitation DEMMI < 20  were more likely to end up with ICU transfer or death in RCC than those with post-rehabilitation DEMMI ≥ 20  (P < 0.001). The duration of rehabilitation was not significantly associated with overall survival. A multivariate Cox proportional hazards model with adjusting for significant covariates showed that weaning success , post-rehabilitation DEMMI ≥ 20 , and higher platelet counts  were significantly associated with better overall survival, while higher total bilirubin level , the presence of end-stage renal disease  and old stroke  were associated with worse overall survival  and higher Glasgow Coma Scale  than the rehabilitation non-responders (post-rehabilitation DEMMI < 20)  . TherefoBesides weaning success and higher post-rehabilitation functional status, our study showed a higher platelet count was independently associated with both survival at hospital discharge and 3 months after RCC discharge. This finding was generally consistent with previous observational studies and meta-analysis , 28. TheThe present study had some limitations. First, this is a single center study with retrospective nature. Second, survival status beyond 3 months after RCC discharge was not obtained. Third, the average age of the population was more than 70 years, which was higher than that in previous PMV studies . Our resFunctional status after pulmonary rehabilitation was independently associated with weaning success, hospital survival, and 3-month overall survival after RCC discharge in patients requiring PMV support. Post-rehabilitation functional status, but not pre-rehabilitation functional status, was a significant prognostic factor associated with weaning success and survival in patients requiring PMV. Physicians, therefore, should not decline or withhold rehabilitation for PMV patients who had poor performance status at initial post-ICU stage. Further studies were warranted to validate our findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Institutional Review Board authority of National Taiwan University Hospital Hsin-Chu Branch (REC: 107-043-E). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.L-TK, S-KL, C-PT, and M-RL participated in the study concept and design. C-PT, Y-FW, P-HT, C-HC, L-YC, and K-LY participated in the collection of data, statistical analysis, interpretation of data, and manuscript draft. S-KL, M-RL, and J-CK participated in manuscript revision. All authors read and approved the final manuscript.S-KL received speaking honoraria from Norvatis, Pfizer, Roche, and Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The presence of aortic regurgitation (AR) in the setting of ventricular septal defect (VSD) has always been a management challenge.This is a retrospective study looking at patients who underwent VSD closure with or without aortic valve intervention between January 1st, 1992 and December 31st, 2014 at the Institute Jantung Negara. This study looked at all cases of VSD and AR, where AR was classified as mild, moderate, and severe, the intervention done in each of this grade, and the durability of that intervention. The interventions were classified as no intervention (NI), aortic valve repair (AVr), and aortic valve replacement (AVR).A total of 261 patients were recruited into this study. Based on the various grades of AR, 105 patients had intervention to their aortic valve during VSD closure. The rest 156 had NI. All patients were followed up for a mean time of 13.9±3.5 years. Overall freedom from reoperation at 15 years was 82.6% for AVr. Various factors were investigated to decide on intervening on the aortic valve during VSD closure. Among those that were statistically significant were the grade of AR, size of VSD, age at intervention, and number of cusp prolapse.We can conclude from our study that all moderate and severe AR with small VSD in older patients with more than one cusp prolapse will need intervention to their aortic valve during the closure of VSD. Early VSD closure have been proposed to prevent the onset of AR or the worsening of the existing AR.Combination of ventricular septal defect (VSD) and aortic regurgitation (AR) due to prolapse of right coronary or, less frequently, non-coronary cusp is known as Laubry-Pezzi syndromeHowever, once an aortic valve deformity is present, surgical closure of VSD alone without intervention to the aortic valve may not be enough to prevent progressive AR. These patients will require aortic valve repair (AVr) or aortic valve replacement (AVR). Aortic valve prolapse and AR are more frequent and severe in patients with delayed surgery, highlighting the importance of early surgical intervention.In patients with VSD and concomitant AR, moderate and severe AR represent a challenging surgical issue. AVR is often associated with major drawbacks.Mechanical prosthetics require long-term anticoagulation therapy and are often limited by the size of the aortic annulus. On the other hand, homografts and bioprosthetic valves have a high rate of early calcification and failure. Therefore, AVr has been an attractive alternative in the treatment of AR instead of AVR. Hence, this study is aimed mainly at looking at the long-term outcome of aortic valve intervention done in patients with syndrome of VSD and AR.To assess the outcome of aortic valve intervention in patients with AR in the setting of VSD.To identify variables that may predict the outcome of AVr and the risk of reoperation in our study population.st, 1992 and December 31st, 2014 at the Institute Jantung Negara (IJN), Kuala Lumpur , were included in this study. The patients were classified into two major groups where in one group the severity of AR was classified as mild, moderate, and severe, and in the other group the type of aortic valve intervention was classified as no intervention (NI), AVr, and AVR. Correlations between the severity of AR and the type of intervention used to address the AR were all analyzed, and the outcomes of each of these were reviewed together with the factors that contributed to them.This is a retrospective study. Patients with VSD and concomitant AR who underwent VSD repair with or without aortic valve intervention between January 1All patients with VSD and concomitant AR who underwent VSD closure with or without intervention to the aortic valve.Patients with other major cardiac anomalies.Patients with congenital syndromes.Patients with other comorbidities who are unfit for VSD closure.Patients with connective tissue disease or aortopathy features.st, 1992 up to December 31st, 2014. Ethics approval to review all the patients’ medical records were obtained for the Ethics Committee of IJN, Kuala Lumpur, in 2012. All patients who underwent VSD closure were identified and their medical records were reviewed. Only those who had concomitant AR were included in this study.The operation theatre logbooks were reviewed from January 1Patients were divided into three groups - mild AR, moderate AR, and severe AR - based on their preoperative echocardiographic findings. Further information regarding aortic valve intervention for AR was obtained from the operative notes. They were further subdivided into three groups based on the intervention performed - AVr, AVR, and NI.Postoperative echocardiographic results were reviewed from patients’ case notes to assess improvement in the grade of AR after intervention (prior to discharge). All the echocardiographic assessment was done transthoracically as, until 1999, we did not have the small probe for transesophageal echocardiography and hence, in order to standardize our findings, we used the transthoracic data to assess all patients. We also noted that there was not much discrepancy of findings among the patients comparing who had transesophageal and who had transthoracic echocardiography after 1999. On follow-up, patients’ AR were quantified based on echocardiographic findings from the medical records. The AR gradient was quantified using pressure half-time measurement. Single measurement was taken for all the VSD. The size was taken as the intraoperative measurement size stated in the operative logbook by the surgeon.Echocardiographic size of the VSD was not taken as with the presence of a prolapsed leaflet, the size measured might not be accurate. Other details about type of VSD, number of prolapsed cusps, type of AVr, cardiopulmonary bypass time, cross-clamp time, postoperative complications, and the need for reoperation were obtained from the operative notes and medical records. We define failure of the intervention if AR is more than moderate on pressure half-time measurement. We also tested all the valves with saline leak test intraoperatively and we accept success of the intervention if the three aortic cusps come together and hold the saline with not much leak into the ventricle.P-value of < 0.05.All data were entered and analyzed using IBM Corp. Released 2011, IBM SPSS Statistics for Windows, Version 20.0, Armonk, NY: IBM Corp. Statistical significance was set at a Preoperative AR grades were divided into three categories: mild, moderate, and severe AR. These categorical variables were compared with other variables using Pearson’s chi-squared test.Differences in parametric variables among those three groups were compared by one-way analysis of variance (or ANOVA).Univariate regression analysis was used to determine factors affecting the outcome of AVr. Multivariate analysis was then performed if there was presence of a predictive factor. Freedom from reoperation was analyzed by using Kaplan-Meier actuarial survival analysis. The log rank test was used for comparisons of Kaplan-Meier freedom from reoperation curves.A total of 261 patients who met the inclusion criteria between January 1992 and December 2014 were included in the study. The mean age at surgery was 10.6±9.3 years, with 60.9% of the patients being male. There were 170 (65%) Malay patients, 56 (21.5%) Chinese patients, 12 (4.6%) Indian patients, and 23 (8.8%) other origin patients.Most of our patients (50.6%) had perimembranous VSD, followed by doubly committed subaortic VSD (46%), and muscular VSD (3.4%). The mean VSD size was 1.2±0.6 cm (range: 0.4 - 3.0 cm). Among these 261 patients with AR in the setting of VSD, 164 (62.8%) had mild AR, 54 (20.7%) had moderate AR, and 43 (16.5%) had severe AR.P=0.002). The preoperative AR grade was also significantly associated with the patients’ age (P=0.022) (P<0.001). Those patients with two or three prolapsed cusps had higher incidence of moderate and severe AR as compared to those with none or one prolapsed cusp, who had mainly mild AR (Further cross tabulation (Pearson’s chi-squared test) revealed that there was significant association between VSD size and preoperative AR grade in this study (P=0.022) . Moreove mild AR .Aortic valve intervention for various grades of AR is shown in P=0.002). Various factors were associated with the decision on the type of aortic valve intervention, especially when dealing with moderate and severe AR. It was shown in this study (P<0.001), size of VSD (P<0.001), grade of preoperative AR (P<0.001), and number of prolapsed cusps (P<0.001).In this study, 84 patients underwent AVr, where 50 out of the 84 (59.5%) patients had Trussler’s repair, followed by commissural-related repair (15.5%), leaflet plication (13.1%), and others (11.9%). It was shown that there is a significant association between the type of repair performed and the number of prolapsed cusps in this study . In this study, it was also noted that the type of aortic valve intervention (AVr/AVR/NI) was significantly affected by the number of prolapsed cusps (P<0.001). Most of patients with three prolapsed cusps had AVR (71.4%) done, while patients with none or one prolapsed cusp had NI done .In terms of age, those of the older age group (≥ 31 years) had a higher percentage of AVR (55.6%), while those in the younger age group (≤ 30 years) had a higher percentage of NI (61.1%) and AVr (32.5%) . We found that younger patients and those who underwent leaflet plication as their repair technique had better outcome compared to others.Univariate analysis was performed to determine the factors influencing the outcome of AVr on follow-up to improve patient selection in the future. Factors such as age, type of VSD, VSD size, preoperative grade of AR, cross-clamp time, cardiopulmonary bypass time, type of AVr, and AR grade immediately after operation, as well as the number of prolapsed cusps, were analyzed . Age andP-values of 0.048 and < 0.001, respectively).Using similar factors, univariate analysis was also performed to determine the factors affecting reoperation . It was A residual moderate or severe AR in the immediate postoperative period has been shown to be the main reason for reoperation in our cohort of patients. Pertaining to VSD size, we also found that the patients with smaller size VSD had higher incidence for reoperation.There was no hospital mortality among these 261 patients. Overall freedom from reoperation at 10 and 15 years were 86.9% and 85.6%, respectively, for AVr, and freedom from reoperation for AVR at 10 and 15 years were 88.9% and 88.9%, respectively.P-value of 0.074). P-value of 0.673). For those who underwent leaflet plication as a method of AVr, freedom from reoperation at 10 and 15 years were 100% in this study. For Trussler’s repair, the freedom from reoperation was 83.8% at 10 years as well as 15 years, while for commissural-related procedures, the freedom from reoperation at 10 and 15 years were 84% and 83.3%, respectively.Among those with NI, freedom from reoperation at 10 and 15 years were 98.0% for both periods. The Kaplan-Meier curve in All patients were followed up for a mean time of 13.9±3.5 years (range: 0.5 - 25 years). Among the 261 patients who were included in this study, 114 (43.7%) of them are still under follow-up, 73 (28%) have been discharged from follow-up, while 52 patients (19.9%) have been transferred to another state hospital for follow-up and 22 (8.4%) patients are lost to follow-up.The patients with moderate and severe AR had longer follow-up . The follow-up duration was shorter in the mild AR group (mean time of 2.9±2.3 years)..This study demonstrates that patients with VSD and concomitant AR have several treatment options. Addressing the aortic valve at an earlier age has been shown to provide a better outcome,3.This is due to the fact that, if left untreated, AR will progress to a more severe form in the older age, necessitating AVR rather than AVr. This is consistent in our study, in which the median age at operation was eight years. Older patients presented with more severe AR (P=0.022). The median age for the AVR group was older (25 years old) while those who underwent AVr were younger (median age of nine years).In most patients, AR tends to be detected between the ages of three to eight years,3 have pointed out that the grade of AR is associated with the size of VSD, and this relationship was also seen in this study . A large VSD has been shown to have a lower AR gradient. The reason for this has been mainly related to the venturi effect that happens to the leaflets. In a smaller VSD defect, the suction effect to the leaflet is far greater than in a larger defect, and this displace the cusp even more and exaggerates the prolapse of the leaflets, hence worsening the AR. We had 15 patients with VSD size of > 2.1 cm; 40% of these patients had mild AR, 33% had moderate AR, while only 27% had severe AR. This showed that the larger the VSD size, the lesser the severity of AR. In a small VSD, the venturi effect is far greater than in larger VSDs. This also will exert more strain to the affected leaflet.Previous reportsConstant pulling effect on the affected leaflet can damage the microarchitecture of the leaflet and this can predispose the leaflet to fail in coming years.. The best method would still be intraoperative measurement of the VSD defect, which was performed in this study.Echocardiography is not very reliable in providing the true size of the VSD, especially in the presence of AR, because the true size tends to be underestimated,7. The ideal management should be one which is safe, simple, reproducible, and durable, and should deal with all the anatomic components of the syndrome, preferably at a young age. In practice, achieving these deceptively simple goals is difficult.Although the syndrome of VSD and AR has been long recognized as a clinical entity, there is still no common agreement about its management, particularly with regards to the indication, timing, and technical details of the surgical procedureIn our series, all patients with a preoperative moderate or severe AR gradient had their aortic valves inspected and assessed during surgery. We had a few criteria which we used to assess the valve on table and this included the effective height of each leaflet, the support from the commissures, symmetrical apposition of the leaflet by using the Nodule of Arientus as a guide, and leaflet appearance. Leaflets which have rolled edges, elongated and asymmetrical, underwent repair even if the AR was only mild. We also had patients with had moderate AR, but with normal cusp and commissures, who had NI to their aortic valve. All this assessment was solely decided by the operating surgeon and this is one of the main limitations of our study as we had no standard criteria to select patients for AVr.In the mild AR group, we had two AVr that failed, both of these using the Trussler’s technique, and we had six patients that had NI in the mild group progressing to more than moderate AR after the VSD was closed. We feel that the Trussler’s repair has its limitations, the patient must be carefully selected, and it is not suitable if there is leaflet elongation. The six patients who had NI to their valve were found to be older children (range: 6 - 8 years old) and all of them had a perimembranous VSD with a size < 1 cm. The moderate AR group had six patients who had NI, and these patients still had a competent aortic valve on their latest follow-up. We feel that as long the leaflet has no major structural abnormality and it has good commissural support, the closure of the VSD itself should be sufficient. This, however, must be an accurate assessment by the operating surgeon.,11 have shown associations between preoperative AR grade and subsequent AR progression as well as the type of intervention. In this study, we further clarify that such association was present between the preoperative AR grade and the progression of the AR in coming years . In addition to the preoperative AR grade, older age at VSD repair (P<0.001) as well as VSD size (P<0.001) also predicted the type of aortic valve intervention. We had 15 patients with mild AR that underwent AVr and this was mainly due to the presence of right coronary cusp prolapse in the doubly committed subarterial group of patients. Although the AR was mild, we decided to plicate the leaflet as it was elongated into the VSD defect as a windsock deformity.Previous studies. This may be the reason why older age at VSD repair predicts the necessity for AVR.In our study, the median age for AVR was 25 years and none of our patients who needed AVR was younger than nine years old. If the VSD shunt persisted and patients grew older, prolapse would get worse with time and even progressed to sinus of Valsalva aneurysm. The more the damage to the aortic valve, the more likely AVR will be needed.Moreover, it has also been reported that the longer the wait for surgery, the more morphological changes happen to the aortic cusps, making AVr less suitable. Therefore, attempts to surgically treat AR are directed towards AVr.AR in patients with VSD is commonly associated with prolapse of the aortic valve cusps, with elongation of the free edgeP=0.001), and therefore, it had a significant association with the type of aortic valve intervention (P<0.001). Besides that, there was also significant association between the type of AVr performed with the number of prolapsed cusps in our study (P=0.002).In this study, the number of prolapsed aortic cusps were significantly associated with the preoperative grade of AR . Those with three cusps prolapsed mainly had an AVR done (71.4%). Replacement of an entire aortic valve is not done when only one cusp is prolapsed.Among the 84 patients who had AVr done, most of them underwent Trussler’s repair (59.5%). The type of AVr employed was significantly associated with the number of prolapsed cusps  and disadvantages of AVr  must be balanced against the outcomes of AVR so that AVr becomes an attractive and justifiable alternativeP-value of 0.048 and < 0.001, respectively).It was also noted in this study that the VSD size as well as the postoperative AR grade significantly affected reoperation (,12.There was no hospital mortality noted in this study period consistent with most other published studiesThis was a retrospective study through a long period. Indications for surgical intervention on the aortic valve have changed, as have AVR options, techniques of repair, and available material. We did not have a large enough group of patients for AVr as well as AVR, which may have affected our outcome. Although we could not identify a significant difference in freedom from reoperation between AVr and AVR, the curves do diverge. It is quite possible that if we had had more patients, this divergence would have reached significance.AVr is an effective and durable technique for the surgical treatment of AR in patients with VSD. In this study, AVr achieved a satisfactory success rate.This stressed the importance of early detection and intervention prior to progression of AR, thus reducing the rate of AVR. However, longer follow-up with larger number of patients would provide a better assessment of the outcome of AVr in patients with syndrome of VSD and AR. highlighted that the youngest patient they operated on was at three months old, and this was also the same in our series. In that series, they also found that patients did better when they were operated at a younger age. A similar finding that we also found in our series. We, however, had a longer follow-up and patient cohort to support this finding in comparison with the recent study.Over the years it has always been a debate to decide on when is it safe to intervene this group of patients. A recent publication. The procedure has been shown to be safe and feasible in the setting of AR. In a recent review for the safety of device closure for VSD, it was found that percutaneous and perventricular device closure for VSD are safe and did not have any extra morbidity.The risk of cardiopulmonary bypass has always been the concern for young infants. There have been recent reports on transcatheter closure of VSD in aortic valve prolapse and ARThe avoidance of cardiopulmonary bypass will further bring down the operative age and further protect the structural damage to the aortic valve. This will enable early VSD closure and probably NI for the aortic valve in coming years.This is a retrospective study and most of the data were collected from the operative notes and patients' medical records. The decision to intervene the valve was solely of the operating surgeon and there was no strict criteria or guideline that was used as a guide for the valve intervention. The actual size of the VSD was also a size that was estimated by the operating surgeon.We can conclude from our study that all moderate and severe AR with small VSD in older patients with more than one cusp prolapse will need intervention to their aortic valve during the closure of the VSD.