Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions.

BACKGROUND: Exosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood. METHODS: Super-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions. RESULTS: A significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion. CONCLUSION: These results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.

Clinicopathological features and outcome in elderly patients with idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy (IMN) is the leading cause of nephrotic syndrome in the elderly. The treatment of idiopathic membranous nephropathy is quite challenging due to the particularity of elderly patients. This study intends to investigate the clinicopathological characteristics and initial therapeutic effect of idiopathic membranous nephropathy among elderly patients. METHODS: A retrospective study of 67 elderly patients (58.2% male, median age 69.0 years, range, 65-83 years) with biopsy-proven membranous nephropathy was conducted at Guangdong Provincial People's Hospital from 2016 to 2020. Data on clinicopathological characteristics and initial therapeutic effects were analyzed. RESULTS: Of the 67 patients, the mean eGFR of overall patients was 66.49 mL/min/1.73m2 while the median urine protein-to-creatinine ratio (uPCR) and urine albumin-to-creatinine ratio (uACR) was 5676.73 mg/g and 2951.56 mg/g, respectively. Pathological data revealed that the membranous Churg's stage II was the most frequent (71.64%). Moreover, glomerular PLA2R antigen fluorescence intensity of (+) and IgG4 antigen fluorescence intensity of (++) were detected in 63.6% and 86.4% of all patients, respectively. Overall, 44 patients, accounting for 65.7%, achieved remission including complete remission and partial remission within 1 year after renal biopsy. Compared with a non-remission group, the levels of uPCR (6274.6 vs. 3235.6 mg/g, p = 0.007) and uACR (3433.6 vs. 1773.2 mg/g, p = 0.017) were significantly higher in remission group. The proportion of immunosuppressive therapy in the remission group was also higher (86.4% vs. 30.4%, p < 0.01). Compared with conservative treatment, patients with combined treatment with glucocorticoid and cyclophosphamide (CTX) or glucocorticoid and calcineurin inhibitor (CNIs) achieved higher remission rate (glucocorticoid plus cyclophosphamide vs. conservative treatment, 84.6% vs. 27.3%, p = 0.001; glucocorticoid plus calcineurin inhibitor vs. conservative treatment, 88.0% vs. 27.3%, p < 0.001). Further analysis showed that compared with patients who underwent conservative treatment, the proportion of males, the levels of uPCR, uACR, BUN, Scr, CysC and PLA2R antigen-positive staining rate in kidney biopsy were higher in those who underwent combined treatment with glucocorticoid and CTX, while the levels of eGFR, TP and ALB were lower (p < 0.05). In addition, patients who received combined treatment with glucocorticoid and CNIs had higher levels of uPCR, uACR, TC and lower levels of TP, ALB than those who received conservative treatment (p < 0.05). Moreover, there were no statistically significant differences in the 1-year progression rate in eGFR between the immunosuppressive treatment group and conservative treatment group (3.3 vs. 0.2 ml/min/1.73m2, p = 0.852). CONCLUSIONS: Most elderly patients diagnosed with IMN had multiple comorbidities, and the membranous Churg's stage II was most common. The glomerular PLA2R and IgG4 antigen deposition were frequently detected accompanied by glomerulosclerosis and severe tubulointerstitial injury. For high-risk elderly patients with severe proteinuria, early initial immunosuppressive therapy could achieve a higher urinary protein remission rate. Therefore, it is crucial for clinicians to balance the risks and benefits of immunosuppressive therapy based on clinical and pathological characteristics and develop individualized treatment regimens for elderly patients with IMN.

Applicability of Creatinine-based equations for estimating glomerular filtration rate in elderly Chinese patients.

BACKGROUND: The accuracy of the estimated glomerular filter rate (eGFR) in elderly patients is debatable. In 2020, a new creatinine-based equation by European Kidney Function Consortium (EKFC) was applied to all age groups. The objective of this study was to assess the appropriateness of the new EKFC equation with Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), Lund-Malmö Revised (LMR), Berlin Initiative Study 1 (BIS1), and full age spectrum (FAS) equations based on serum creatinine (SCR) for elderly Chinese patients. METHODS: A total of 612 elderly patients with a measured glomerular filtration rate (mGFR) by the dual plasma sample clearance method with Technetium-99 m-diethylenetriamine-pentaacetic acid (Tc-99 m-DTPA) were divided into four subgroups based on age, sex, mGFR, and whether combined with diabetes. The performance of GFR was assessed while considering bias, precision, accuracy, and root-mean-square error (RMSE). Bland-Altman plots, concordance correlation coefficients (CCCs), and correlation coefficients were applied to evaluate the validity of eGFR. RESULTS: The median age of the 612 participants was 73 years, and 386 (63.1%) were male. Referring to mGFR (42.1 ml/min/1.73 m2), the CKD-EPI, LMR, BIS1, FAS, and EKFC equations estimated GFR at 44.4, 41.1, 43.6, 41.8 and 41.9 ml/min/1.73 m2, respectively. Overall, the smallest bias was found for the BIS1 equation (- 0.050 vs. range - 3.015 to 0.795, P<0.05, vs. the CKD-EPI equation). Regarding P30, interquartile range (IQR), RMSE, and GFR category misclassification, the BIS1 equation generally performed more accurately than the other eqs. (73.9%, 12.7, 12.9, and 35.3%, respectively). Nevertheless, no equation achieved optimal performance for the mGFR≥60 ml/min/1.73 m2 subgroup. Bland-Altman analysis showed the smallest mean difference (- 0.3 ml/min/1.73 m2) for the BIS1 equation when compared to the other equations. CONCLUSIONS: This study suggested that the BIS1 equation was the most applicable for estimating GFR in Chinese elderly patients with moderate to severe renal impairment.

Comparison of estimated glomerular filtration rate equations based on serum creatinine-, cystatin C- and creatinine-cystatin C in elderly Chinese patients.

BACKGROUND: This study aimed to further evaluate the accuracy of eleven GFR equations in different subgroups of an elderly Chinese hospitalized population. METHODS: All participants of the study were divided into seven separate groups including age-subgroup, sex-subgroup, GFR Staging-subgroup and whether combined with diabetic, hypertensive, coronary heart disease (CHD) and cerebrovascular disease. Referring to Tc-99m-DTPA dual plasma sample clearance method, six serum creatinine (Cr)-based [Cockcroft-Gault (CG), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPICr), Lund-Malmö Revised (LMR), Berlin Initiative Study (BIS1), Full Age Spectrum (FASCr) and European Kidney Function Consortium (EKFC)], two serum cystatin C(Cys)-based (CKD-EPICys and FASCys), and three Cr-Cys combination based (CKD-EPICr-Cys, BIS2 and FASCr-Cys) equations were employed. Bias, interquartile range of the median difference (IQR), P30, and GFR misclassification rate were calculated to compare the performance of the selected equations. RESULTS: A total of 359 elderly Chinese patients were enrolled. Overall, median mGFR was 36.91(25.26,56.32)ml/min/1.73 m2. Smaller biases (ml/min/1.73 m2) were shown in CKD-EPICr and BIS1 equations (0.75 and 0.61). IQR (ml/min/1.73m2) was least with BIS2 equation and FASCr-Cys equation (10.34 and 10.65). For accuracy (P30), performance of FASCr-Cys, BIS2, and BIS1 equation was superior (78.3%, 78.0%, and 74.7%, respectively). In terms of RMSE (ml/min/1.73 m2), BIS1 and FASCr-Cys equation performed better (12.44 and 12.51). CONCLUSIONS: Overall, this study showed that the eGFR equations were less accurate in the diabetic and non-hypertension group than in the non-diabetic and hypertension group, respectively. Among all enrolled equations, the BIS2 and FASCr-Cys equations might be the best choice to evaluate glomerular filtration rate in Chinese elderly patients.

Cardiac cell type-specific responses to injury and contributions to heart regeneration.

Heart disease is the leading cause of mortality worldwide. Due to the limited proliferation rate of mature cardiomyocytes, adult mammalian hearts are unable to regenerate damaged cardiac muscle following injury. Instead, injured area is replaced by fibrotic scar tissue, which may lead to irreversible cardiac remodeling and organ failure. In contrast, adult zebrafish and neonatal mammalian possess the capacity for heart regeneration and have been widely used as experimental models. Recent studies have shown that multiple types of cells within the heart can respond to injury with the activation of distinct signaling pathways. Determining the specific contributions of each cell type is essential for our understanding of the regeneration network organization throughout the heart. In this review, we provide an overview of the distinct functions and coordinated cell behaviors of several major cell types including cardiomyocytes, endocardial cells, epicardial cells, fibroblasts, and immune cells. The topic focuses on their specific responses and cellular plasticity after injury, and potential therapeutic applications.

A comparative study of four Bowie-Dick test under the condition of pressure steam sterilizer simulating gas leakage.

We aimed to understand the evaluation of different Bowie-Dick test (B-D test) on the performance of pressure steam sterilization equipment in the case of simulated gas leakage, and we selected a pulsating vacuum steam sterilizer to set 4 different gas leakage levels: 1.1, 1.3, 1.5, and 1.7 mbar/min during the B-D test phase. In terms of methods, 4 different brands of B-D test kits (devices) were tested at 4 different leakage rates, and a total of 48 experiments were conducted. The results from univariate analysis revealed that there are statistically significant differences in the judgment of test results among different personnel and brands. The results from multivariate logistic regression analysis displayed that the difference between different personnel was statistically significant (χ = 45.34, P < .001); the difference between different products was statistically significant (χ = 129.37, P < .001); and there was no statistically significant difference between different degree of leakage (χ = 6.99, P > .05). Result judgments of brand 1 and brand 2 are susceptible to subjective factors. The judgment of brand 3 is intuitive and consistent with the evaluation result of brand 4. In conclusion, the order of capacity to evaluate air leakage from best to worst is brand 4→brand 3→brand 1→brand 2.

Advancing Image Understanding in Poor Visibility Environments: A Collective Benchmark Study.

Existing enhancement methods are empirically expected to help the high-level end computer vision task: however, that is observed to not always be the case in practice. We focus on object or face detection in poor visibility enhancements caused by bad weathers (haze, rain) and low light conditions. To provide a more thorough examination and fair comparison, we introduce three benchmark sets collected in real-world hazy, rainy, and low-light conditions, respectively, with annotated objects/faces. We launched the UG2+ challenge Track 2 competition in IEEE CVPR 2019, aiming to evoke a comprehensive discussion and exploration about whether and how low-level vision techniques can benefit the high-level automatic visual recognition in various scenarios. To our best knowledge, this is the first and currently largest effort of its kind. Baseline results by cascading existing enhancement and detection models are reported, indicating the highly challenging nature of our new data as well as the large room for further technical innovations. Thanks to a large participation from the research community, we are able to analyze representative team solutions, striving to better identify the strengths and limitations of existing mindsets as well as the future directions.

Comprehensive chemical profiling of monascus-fermented rice product and screening of lipid-lowering compounds other than monacolins.

ETHNOPHARMACOLOGICAL RELEVANCE: Monascus-fermented rice product (MFRP) has been regarded as a dietary supplement and traditional medicine with circulation-promoting effects in China and other countries for centuries. AIM OF THE STUDY: This study was carried out to profile the chemical components in MFRP, and provide available information for elucidating the potential lipid-lowering compounds other than monacolins. MATERIALS AND METHODS: High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) and gas chromatography coupled with mass spectrometry (GC-MS) methods were applied to comprehensive analysis of chemical components in MFRP. Potential small molecules were identified by comparing with reference standards, or tentatively characterized by comparing their retention time and high-resolution mass spectral data with previous literature. The lipid-lowering properties of ten major non-monacolin compounds were evaluated in cholesterol-fed zebrafish larvae. And one with optimum lipid-lowering activity was subsequently evaluated in high fat diet-fed C57BL/6 J mice, with the dyslipidemia and ectopic lipid deposition being investigated. RESULTS: A total of 99 compounds were characterized in MFRP, including 38 monacolins, 5 decalins, 6 isoflavones, 13 pigments, 8 azaphilonoids, 11 amino acids, 4 nucleosides, 9 lipid acids, 4 phytosterols and glycerol. The preliminary screening showed that ergosterol remarkably reduced cholesterol levels in zebrafish larvae. Moreover, ergosterol delayed body weight gain and decreased circulating total cholesterol, triglyceride, low density lipoprotein cholesterol levels in high fat diet-fed mice. Ectopic lipid accumulation was also ameliorated in the liver and heart of obese mice. CONCLUSION: Global analysis of chemical components and screening of lipid-lowering non-monacolin compounds in MFRP have improved our understanding of its therapeutic material basis.

Integration of Multiple Analytical and Computational Tools for the Discovery of High-Potency Enzyme Inhibitors from Herbal Medicines.

Herbal medicines (HMs) are an important source of drugs. In this study, an efficient strategy integrating ultrafiltration LC-MS, microplate bioassays, and molecular docking was proposed to screen high-potency enzyme inhibitors from HMs. Using this strategy, the structure-activity relationships (SARs) including binding-affinity-based SAR, enzymatic-activity-based SAR, and structural-complementarity-based SAR of compounds in an HM can be analyzed to provide abundant information for drug discovery. A prominent advantage of the approach is that it offers a multidimensional perspective to understand enzyme-ligand interactions, which could help to avoid false-positive screening results brought by a single method. By using xanthine oxidase (XOD) as an illustrative case, two types of potent XOD inhibitors, including flavones and coumarins, were successfully screened out from an HM of Ginkgo biloba. The study is expected to set a solid foundation for multidisciplinary cooperation in drug discovery.