Circ-USP9X interacts with EIF4A3 to promote endothelial cell pyroptosis by regulating GSDMD stability in atherosclerosis.

Endothelial pyroptosis is a pathological mechanism of atherosclerosis (AS). Circular RNAs (circRNAs) are vital in AS progression by regulating endothelial cell functions. The study aimed to explore whether circ-USP9× regulated pyroptosis of endothelial cell to involve in AS development and the molecular mechanism. Pyroptosis was determined using lactate dehydrogenase (LDH) assay, enzyme linked immunosorbent assay (ELISA), flow cytometry, propidium iodide (PI) staining assay, and western blot. The mechanism of circ-USP9× was determined using RNA pull-down and RNA binding protein immunoprecipitation (RIP) assays. Results showed that circ-USP9× was upregulated in AS and oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). Knockdown of circ-USP9× suppressed ox-LDL induced pyroptosis of HUVECs. Mechanically, circ-USP9× could bind to EIF4A3 in the cytoplasm. Moreover, EIF4A3 was bound to GSDMD and further affects GSDMD stability. Overexpression of EIF4A3 rescued cell pyroptosis induced by circ-USP9× depletion. In short, circ-USP9× interacted with EIF4A3 to enhance GSDMD stability, thus further promoting ox-LDL-induced pyroptosis of HUVECs. These findings suggested that circ-USP9× participates in AS progression and may be a potential therapeutic target for AS.

Genome-Wide Identification of BES1 Gene Family in Six Cucurbitaceae Species and Its Expression Analysis in Cucurbita moschata.

The BES1 (BRI1-EMSSUPPRESSOR1) gene family play a vital role in the BR (brassinosteroid) signaling pathway, which is involved in the growth and development, biotic, abiotic, and hormone stress response in many plants. However, there are few reports of BES1 in Cucurbita moschata. In this study, 50 BES1 genes were identified in six Cucurbitaceae species by genome-wide analysis, which could be classified into 3 groups according to their gene structural features and motif compositions, and 13 CmoBES1 genes in Cucurbita moschata were mapped on 10 chromosomes. Quantitative real-time PCR analysis showed that the CmoBES1 genes displayed differential expression under different abiotic stress and hormone treatments. Subcellular localization showed that the most of CmoBES1 proteins localized in nucleus and cytoplasm, and transactivation assay indicated 9 CmoBES1 proteins played roles as transcription factors. Our analysis of BES1s diversity, localization, and expression in Curcubitaceae contributes to the better understanding of the essential roles of these transcription factors in plants.

Honeycomb-like MnO2/Biochar Catalyst Fabricated by High-Energy Electron Beam Irradiation for Degradation of Antibiotics in Swine Urine.

The modification of biochar is essential for the development of multifunctional biochar materials with enhanced remediation effects on contaminated water. In this work, a biochar-based microcatalyst with sunlight sensitivity was synthesized by a creative modification method that involved the rapid fabrication of MnO2 microspheres by high-energy electron beam (HEEB) irradiation, and loading them into corn straw-derived honeycomb-like KOH-modified biochar (MBC) to obtain a sunlight-sensitive microcatalyst (SSM). The honeycomb-like structure of MBC facilitated the improvement in MnO2 dispersion and photocatalytic property through confinement effect. The effects of photocatalyst dosage, initial chlortetracycline (CTC) concentration, solution pH, temperature and coexisting ions on the photocatalytic performance of SSM were systemically investigated. The results indicated that SSM could efficiently degrade CTC in water and swine urine under sunlight, and exhibited high stability against coexistence of urea, Cl- and SO42-. Moreover, SSM showed good reusability in regeneration studies. This work provides a novel method for degrading CTC with potential application prospect.

Efficient Simultaneous Detection of Metabolites Based on Electroenzymatic Assembly Strategy.

Objective and Impact Statement: We describe an electroenzymatic mediator (EM) sensor based on an electroenzymatic assembly peak separation strategy, which can efficiently realize the simultaneous detection of 3 typical cardiovascular disease (CVD) metabolites in 5 µl of plasma under one test. This work has substantial implications toward improving the efficiency of chronic CVD assessment. Introduction: Monitoring CVD of metabolites is strongly associated with disease risk. Independent and time-consuming detection in hospitals is unfavorable for chronic CVD management. Methods: The EM was flexibly designed by the cross-linking of electron mediators and enzymes, and 3 EM layers with different characteristics were assembled on one electrode. Electrons were transferred under tunable potential; 3 metabolites were quantitatively detected by 3 peak currents that correlated with metabolite concentrations. Results: In this study, the EM sensor showed high sensitivity for the simultaneous detection of 3 metabolites with a lower limit of 0.01 mM. The linear correlation between the sensor and clinical was greater than 0.980 for 242 patients, and the consistency of risk assessment was 94.6%. Conclusion: Metabolites could be expanded by the EM, and the sensor could be a promising candidate as a home healthcare tool for CVD risk assessment.

Heterogeneous ozonation of ofloxacin using MnOx -CeOx /γ-Al2 O3 as a catalyst: Performances, degradation kinetics and possible degradation pathways.

In this study, the performance of ofloxacin (OFX) degradation in synthetic wastewater using synthesized MnOx -CeOx /γ-Al2 O3 as a heterogeneous ozonation catalyst was evaluated. The removal rates of OFX and chemical oxygen demand (COD) during 15-day continuous-flow experiments were 98.2% and 76.7% on average, respectively. An ozone index (mgCOD/mgO3 ) of 1.09 with a high ozone utilization efficiency of 91.39% was achieved. The pseudo-first-order rate constant of ofloxacin degradation reached 15.216 × 10-2  min-1 , which was five times that (3.085 × 10-2  min-1 ) without catalysts. The results of gas chromatography-mass spectrometry (GC-MS) demonstrated that a variety of small-molecule organics occurred in the final oxidation products, such as 4-hydroxyl-4-methyl-2-pentanone and 2-oxoadipic acid in addition to homologs of OFX. The results of this study suggested that hydroxyl radicals played critical roles in the degradation and mineralization of OFX via four main pathways: (a) electrophilic addition of nitrogen; (b) breakdown of carbon-carbon double bonds; (c) hydrolysis of ether rings; and (d) halodecarboxylation of carboxyl groups. The biodegradability (BOD5 /COD) of OFX after catalytic ozonation reached 0.54. PRACTITIONER POINTS: Ofloxacin wastewater was treated using catalytic ozonation in a 15-day continuous experiment with MnOx -CeOx /γ-Al2 O3 as a catalyst. The ozone index reached 1.09 mgCOD/mgO3 during ozonation of ofloxacin. The presence of the catalyst increased the reaction rate constant by a factor of five. 4-hydroxy-4-methyl-2-pentanone was the primary ofloxacin oxidation product.

Blood pressure assessment with in-ear photoplethysmography.

Objective. In this study, we aimed to estimate blood pressure (BP) from in-ear photoplethysmography (PPG). This novel implementation provided an unobtrusive and steady way of recording PPG, whereas previous PPG measurements were mostly performed at the wrist, finger, or earlobe.Methods. The time between forward and reflected PPG waves was very short at the ear site. To minimize errors introduced by feature extraction, a multi-Gaussian decomposition of in-ear PPG was performed. Both hand-crafted and whole-based features were extracted and the best combination of features was selected using a backward-search wrapper method and evaluated by the Akaike information criteria. Hemodynamic parameters such as compliance and inertance were estimated from a four-element Windkessel (WK4) model, which was used to pre-classify PPG signals and generate different BP estimation algorithms. Calibration was done by using previous measurements from the same class. To validate this novel approach, 53 subjects were recruited for a one-month follow-up study, and 17 subjects were recruited for a two-month follow-up study. Calibrated systolic BP estimation accuracy was significantly improved with inertance-based pre-classification, while diastolic BP showed less improvement.Results. With proper feature selection, pre-classification and calibration, we have achieved a mean absolute error of 5.35 mmHg for SBP estimation, compared to 6.16 mmHg if no pre-classification was carried out. The performance did not deteriorate in two months, showing a decent BP trend-tracking ability.Conclusion. The study demonstrated the feasibility of in-ear PPG to reliably measure BP, which represents an important technological advancement in terms of unobtrusiveness and steadiness.

Circular RNA circ_0090231 promotes atherosclerosis in vitro by enhancing NLR family pyrin domain containing 3-mediated pyroptosis of endothelial cells.

Atherosclerosis (AS) is an inflammatory disease caused by multiple factors. Multiple circRNAs are involved in the development of AS. The present study focusses on delineating the role of circ_0090231 in AS. Human aortic endothelial cells (HAECs) were treated with oxidized low-density lipoprotein (ox-LDL) to construct an in vitro AS model. Real-time quantitative polymerase-chain reaction (RT-qPCR) was used to detect the levels of circ_0090231, IL-1ß, and IL-18 transcripts. CircRNA/target gene interactions were predicted using StarBase and TargetScan and confirmed using an RNA pull-down assay and dual-luciferase reporter assay. Further, 3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide (MTT) and lactate dehydrogenase (LDH) release assays were performed to evaluate cell viability and damage in the AS model, respectively. Cell pyroptosis and protein expression were determined using flow cytometry and western blotting respectively. The treatment of HAECs with ox-LDL not only led to significant increase in the levels of circ_0090231 but also resulted in improved cell viability as well as reduced cell injury and pyroptosis as compared to that in non-treated cells. The circ_0090231 was also identified to function as a sponge for miR-635, knockdown of which reverses the effects of circ_0090231 inhibition. Furthermore, our results revealed that levels of NLRP3, a miR-635 target, are not only augmented in the AS model but its overexpression also weakens the miR-635 regulatory effects in the AS development. Taken together, the circ_0090231/miR-635/NLRP3 axis affects the development of AS by regulating cell pyroptosis, thus providing new insights into the mechanism of AS development.

Lentivirus-mediated RNAi targeting CREB binding protein attenuates neointimal formation and promotes re-endothelialization in balloon injured rat carotid artery.

BACKGROUND/AIMS: Lentiviral vectors provide a promising strategy for the treatment of cardiovascular diseases, owing to their ability to govern efficient and durable gene transfer. However, relatively few studies have been addressed on restenosis after balloon or stent associated arterial injury. We previously found that CREB binding protein (CBP), a powerful transcriptional coactivator, regulated cell proliferation and apoptosis in vascular endothelial and smooth muscle cells. Therefore, we investigated whether inhibition of CBP by lentivirus-mediated small interfering RNA can reduce neointimal formation after arterial injury. METHODS: The carotid arteries from Sprague-Dawley rats were injured by balloon catheter, followed by incubating with 100 microl lentivirus expressing CBP or negative control (NC)-specific short hairpin RNAs (shRNAs) or PBS solution for 30 minutes. The rats were euthanized for real-time PCR, Western blot, immunohistochemical staining, and morphometric analysis at 4 weeks after balloon injury and in vivo gene transfer. RESULTS: Lentiviral shRNA targeting CBP markedly reduced CBP expression. Moreover, CBP siRNA showed potent inhibition on balloon injury-induced Nuclear factor kappaB (NF-kappaB) acetylation. Compared with controls, the significant decrease of neointimal formation by CBP siRNA was accompanied by reduced cell proliferation in the neointima of injured arteries. However, no changes in medial area were observed among these different groups. Interestingly, endothelial cell marker CD31 immunostaining and morphometric analysis both showed that CBP knockdown significantly accelerated re-endothelialization. CONCLUSIONS: These findings suggest that CBP is involved in the control of neointimal formation and re-endothelialization via regulating NF-kappaB acetylation. Lentivirus-mediated CBP silencing may represent a novel therapeutic approach for the prevention of restenosis after vascular interventions.

CBP knockdown inhibits angiotensin II-induced vascular smooth muscle cells proliferation through downregulating NF-kB transcriptional activity.

CREB binding protein (CBP), a powerful transcriptional co-activator for various transcriptional factors, regulates cell behavior in many cell types. Angiotensin II (Ang II) contributes to vascular lesion by promoting vascular smooth muscle cells (VSMCs) proliferation and migration. Therefore, we examined whether CBP knockdown could suppress Ang II-induced VSMCs proliferation, and elucidated its underlying molecular mechanism. We constructed lentiviral vector expressing CBP-specific short hairpin RNAs (shRNAs) that efficiently silenced CBP. VSMCs proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assay. Protein and mRNA expression of CBP and relevant cytokines were examined by Western blot, ELISA, and real-time PCR, respectively. We also used luciferase reporter gene and electrophoretic mobility shift assay (EMSA) to detect Nuclear factor kappaB (NF-kB) transcriptional activity and DNA binding. Meanwhile, NF-kB p65 subunit nuclear translocation was confirmed by immunoblotting. Lentiviral-mediated CBP-shRNAs at different multiplicities of infection (MOI = 100, 150) both significantly suppressed Ang II-induced CBP expression. Knockdown of CBP markedly inhibited Ang II-stimulated VSMCs proliferation and cytokines (TNF-alpha and IL-6) production. However, this inhibitory effect was not enhanced at MOI of 150 compared with MOI of 100 (P > 0.05). CBP siRNA showed the potent inhibition on Ang II-induced NF-kB transcriptional activity. Similarly, no significant difference was found between CBP siRNA lentivirus treatment groups. Furthermore, CBP gene silencing had no effect on NF-kB nuclear translocation and DNA binding. These findings suggest that CBP knockdown inhibits Ang II-induced VSMCs proliferation and the mechanism is involved with downregulation of NF-kB transcriptional activity, not through reduction in NF-kB nuclear translocation or DNA binding. Maintaining proper CBP level may be a potential therapeutic target for Ang II-induced cardiovascular disorders.

cAMP-Response-element-binding-protein-binding protein silencing inhibits thrombin-induced endothelial progenitor cell migration via downregulation of CXCR4 expression.

Previous studies have demonstrated that activation of thrombin receptor could promote endothelial progenitor cell (EPC) migration. As cAMP-response-element-binding-protein-binding protein (CBP) is involved in many cellular biological processes, we hypothesized that CBP mediates thrombin-induced EPC migration. In this study, we examined whether CBP silencing would affect EPC migration induced by thrombin using small interference RNA approach. EPC isolated from the bone marrow of femurs and tibias of Sprague-Dawley rats were cultured and identified, and then were treated by thrombin alone or combined with CBP-shRNA lentivirus. Transwell chamber assay was performed to measure EPC migration. Quantitative real-time polymerase chain reaction and Western blot were carried out to detect the expression of CBP and CXCR4. Thrombin induced CBP expression in a time- and dose-dependent manner. Small interference RNA for CBP downregulated thrombin-induced CBP expression. Thrombin-induced EPC migration was also attenuated by CBP downregulation. Western blot indicated that CXCR4 expression on EPC is upregulated by thrombin and this effect was blocked by CBP silencing. In conclusion, thrombin-induced EPC migration was inhibited by CBP silencing via downregulation of CXCR4 expression, indicating that CBP plays an important role in thrombin-induced EPC migration.

Urotensin II induces migration of endothelial progenitor cells via activation of the RhoA/Rho kinase pathway.

Urotensin II (UII) is a vasoactive peptide with many potent effects in the cardiorenovascular system and may be involved in the pathogenesis of atherosclerosis. Cardiovascular risk factors are often accompanied by reduced numbers of endothelial progenitor cells (EPCs) and their impaired migratory capacity. However, the role of UII in the migration of EPCs has not been reported so far. The aim of this study was to investigate whether UII influences the chemotactic function of bone marrow-derived EPCs and the possible signaling mechanisms involved. As a ligand for the orphan G-protein coupled receptor 14 (GPR14, UT receptor), UII exerts vasoactive functions through activation of the RhoA/Rho kinase pathway. We therefore analyzed the expression of GPR14 mRNA and protein, the activation of RhoA kinase and the phosphorylation of myosin light chain (MLC) in EPCs, isolated from the rat bone marrow. EPCs of 1-4 passages expressed GPR14 mRNA and protein. Chemotaxis assays were performed using Transwell cell-culture chambers with UII (10(-10)-10(-6) M), showing that UII induced chemotaxis of EPCs in a concentration-dependent manner after 3-h treatment (all p < 0.05), with the highest value (about 3-fold increase) at 10(-8) M. UII caused rapid activation of RhoA and increased phosphorylation of MLC. Conversely, a Rho-kinase inhibitor Y-27632 prevented the UII-induced migration and the phosphorylation of MLC. In conclusion, GPR14/UT receptor is expressed in EPCs, and UII induces migration of EPCs via activation of the RhoA/Rho kinase pathway. These findings provide new insights into the actions of UII in atherosclerosis.

Integration of catalytic ozonation and adsorption processes for increased efficiency of textile wastewater treatment.

Advanced and optimized textile wastewater treatment by catalytic ozonation and activated carbon (AC) adsorption was investigated. Scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy indicated that Mn and Ce oxides were successfully loaded on the γ-Al2 O3 support, and MnO2 , Mn2 O3 , CeO2 , and Ce2 O3 were the main components of the catalyst. Actual textile wastewater from biochemical effluent was used as experiment wastewater. The removal efficiencies of chemical oxygen demand (COD) and chromaticity were approximately 30.6% (414-287 mg/L on average) and 99.3% (4,033 times to 27 times on average), respectively during the 30-day on-site continuous-flow test with an ozone dosage, contact time, and gas-liquid ratio of 100 mg/L, 15.7 min, and 2.9, respectively. Following 1 g/L AC adsorption, the effluent COD concentration was reduced to 40 mg/L. By contrast, AC adsorption without catalytic ozonation as pretreatment required 10 g/L AC dosage to achieve similar treatment results. Gas chromatography-mass spectrometry analyses indicated that volatile phenols, sulfides, and aniline in wastewater were completely removed after treatment. Inductively coupled plasma results further showed that the active components of MnOx -CeOx in the catalyst were stable after continuous use for 60 days. PRACTITIONER POINTS: Mesoporous catalyst synthesized by impregnating MnOx -CeOx on γ-Al2 O3 support. Catalytic ozonation and AC adsorption were combined to degrade organics. Maximum degradation of COD and chromaticity by optimizing process variables. The efficiency of the method was compared to that of single AC adsorption.

Slit3 regulates migration of endothelial progenitor cells by activation of the RhoA/Rho kinase pathway.

Nerves and blood vessels are in close proximity, indicating possible biomolecular interactions. Slit/Robo signaling pathways play critical roles in cell proliferation and motility. Endothelial progenitor cells (EPCs) participate in angiogenesis and vascular homeostasis. EPC migration induced by Slit3 has not been fully characterized. Thus, the expression of Slit and Robo in EPCs was examined, and the chemotactic functions of Slit3 and the Slit/Robo signaling pathway regulatory mechanisms were explored. We observed that EPCs express mainly the Robo4 receptor, and its ligand Slit3 plays roles in regulation of EPCs migration through activating the RhoA/Rho related kinases. Regulation of Slit3/-Robo4 signaling in EPCs may provide a new therapeutic target for ischemic disease.

Neuropilin 1 ameliorates electrical remodeling at infarct border zones in rats after myocardial infarction.

BACKGROUND: Electrical remodeling at infarct border zone (IBZ) has been shown to contribute to the occurrence of ventricular arrhythmias after myocardial infarction (MI). Sema3A has been demonstrated to reduce the inducibility of ventricular arrhythmias. Neuropilin 1 (NRP1) is the receptor of Sema3A. In the present study, we investigated whether treatment with NRP1 can ameliorate electrical remodeling at IBZ after MI. METHODS AND RESULTS: Wistar rats underwent sham operation (n = 20), the ligation of left coronary artery (MI group, n = 30), MI with control adenovirus (Ad group, n = 30), and MI with NRP1 adenovirus (NRP1 group, n = 30). Eight weeks after treatment, electrophysiological properties including heart rate variability (HRV), monophasic action potential duration (MAPD), effective refractory period (ERP) and the inducibility of ventricular arrhythmias and the expression of arrhythmia-related ion channel proteins including Kv4.2, Kv4.3, KChIP2 and Kir2.1 at the IBZ of the left ventricle were examined. Compared with the MI or Ad group, NRP1 significantly increased HRV and shortened MAPD and ERP (all P < 0.05). Inducibility of VT by electrophysiological study was significantly lower in the NRP1 group than in the MI or Ad group (P < 0.05). The expression levels of Kv4.2, Kv4.3, KChIP2 and Kir2.1 proteins were significantly decreased in MI group and Ad group. In contrast, the expression levels of these proteins were restored in NRP1 group, which may represent the molecular basis of the NRP1-mediated inhibition of electrical remodeling. CONCLUSIONS: NRP1 can ameliorate electrical remodeling at IBZ after MI.

Urotensin II promotes the proliferation of endothelial progenitor cells through p38 and p44/42 MAPK activation.

Urotensin II (UII) is a vasoactive peptide with many potent effects in the cardiorenovascular system and is also possibly involved in the pathogenesis of atherosclerosis. Endothelial progenitor cells (EPCs) are involved in angiogenesis and vascular homeostasis and may be important in the maintenance of endothelial integrity. The aim of this study was to investigate whether UII has an effect on the proliferation of bone marrow-derived EPCs and the possible signaling mechanisms involved. Bone marrow-derived EPCs were isolated from male Sprague-Dawley rats and cultured in medium containing 5% fetal bovine serum. Cells were incubated with UII for 24 h. The proliferation of EPCs was analyzed by MTT assay. Western blotting was performed to determine the phosphorylation levels of mitogen-activated protein kinases (MAPKs). The results demonstrated that UII promoted the proliferation of EPCs in a concentration-dependent manner in a certain range, and the proliferation was largely suppressed by inhibitors of GPR14 and MAPKs (p38 and p44/42). UII significantly increased the phosphorylation levels of p38MAPK and p44/42MAPK, and these effects were significantly inhibited by respective inhibitors. These findings indicate that UII promotes the proliferation of rat bone marrow-derived EPCs through a process that involves MAPK activation, and provides novel insights regarding the role of UII in the EPC-mediated repair of atherosclerotic injury.

Acute effects of ganglionated plexi ablation on sinoatrial nodal and atrioventricular nodal functions.

Ganglionated plexus (GP) ablation has been shown effective for eliminating atrial fibrillation (AF), the most common clinical tachyarrhythmia. However, the safety of destroying the main cardiac autonomic structures remains unclear. This study investigated the acute effects of GP ablation on the sinoatrial nodal (SAN) and atrioventricular nodal (AVN) functions in a canine model. In 10 open-chest dogs, multiple electrode catheters were sutured at both atria for recording and pacing. SAN and AVN function were evaluated. GP ablation caused no significant change of sinus rate immediately after GP ablation compared with the baseline state. After GP ablation, the sinus node recovery time (SNRT) and corrected SNRT did not show significant changes at long pacing cycle lengths (CLs), and only showed significant decrease at shorter pacing CLs. The AH interval at different pacing CLs, the Wenckebach atrioventricular block (AVB) CL, 2:1 AVB CL or the ventricular rate during AF were not significantly altered by GP ablations. Vagal suppression of SAN and AVN functions was eliminated by GP ablation. GP staining showed abundant choline acetyl transferase or tyrosine hydroxylase positive neurons. These findings suggest the functions of the SAN and AVN are mainly retained after GP ablation. These results may be partially related to destroying both parasympathetic and sympathetic elements in the GP by ablation.