An intense cathodic electrochemiluminescence from carbon-nanosheets in situ grown on glassy carbon electrode and application in immunoanalysis via biometallization strategy.

An intense cathodic electrochemiluminescence (ECL) is reported from a polarized glassy carbon electrode (GCE) in peroxydisulfate solution. After the polarization in 1 M Na2SO4 at the potential of - 3.7 V for 3 s, carbon nanosheets (C-NSs) were in situ grown on the surface of the GCE. Measured in 100 mM K2S2O8 solution, the ECL intensity of the GCE/C-NSs is 112-fold that of a bare GCE. The ECL spectrum revealed that the true ECL luminophore in the GCE/C-NSs-peroxydisulfate system is O2/S2O82- which is promoted by C-NSs. When Cu2+ was electrochemically enriched and reduced to Cu(0) on the catalytic sites of C-NSs, the ECL from GCE/C-NSs/Cu in K2S2O8 solution was decreased with increasing logarithmic concentration of Cu2+ in the range from 10 pM to 1 µM, with a limit of detection (LOD) of 3 pM. An immunoanalysis method is proposed via a biometallization strategy using CuS nanoparticles as the tags and carcinoembryonic antigen (CEA) as the model analyte. After the immune recognition in the microplate, the CuS tags in the immunocomplex were dissolved and the resultant Cu2+ was electrochemically enriched and reduced on the catalytic sites of C-NSs, quenching the ECL intensity of GCE/C-NSs-O2/S2O82- system. The proposed ECL immunoanalysis method was used to quantify CEA in actual serum samples with an LOD of 1.0 fg mL-1, possessing the advantages of simple electrode modification, high sensitivity and good reproducibility.

Gasdermin-E-mediated pyroptosis drives immune checkpoint inhibitor-associated myocarditis via cGAS-STING activation.

Immune checkpoint inhibitor (ICI)-induced myocarditis involves intensive immune/inflammation activation; however, its molecular basis is unclear. Here, we show that gasdermin-E (GSDME), a gasdermin family member, drives ICI-induced myocarditis. Pyroptosis mediated by GSDME, but not the canonical GSDMD, is activated in myocardial tissue of mice and cancer patients with ICI-induced myocarditis. Deficiency of GSDME in male mice alleviates ICI-induced cardiac infiltration of T cells, macrophages, and monocytes, as well as mitochondrial damage and inflammation. Restoration of GSDME expression specifically in cardiomyocytes, rather than myeloid cells, in GSDME-deficient mice reproduces ICI-induced myocarditis. Mechanistically, quantitative proteomics reveal that GSDME-dependent pyroptosis promotes cell death and mitochondrial DNA release, which in turn activates cGAS-STING signaling, triggering a robust interferon response and myocardial immune/inflammation activation. Pharmacological blockade of GSDME attenuates ICI-induced myocarditis and improves long-term survival in mice. Our findings may advance the understanding of ICI-induced myocarditis and suggest that targeting the GSDME-cGAS-STING-interferon axis may help prevent and manage ICI-associated myocarditis.

Wavelength-dependent photoelectrochemical response demonstrated by the determination of acetaminophen and rutin in differential molecularly imprinted polymers strategy.

Herein, the excitation wavelength-dependent responses of the molecularly imprinted polymer (MIP) photoelectrochemical (PEC) sensors were investigated, using acetaminophen (AP), rutin (RT) and perfluorooctanoate (PFOA) as the model templates, pyrrole as functional monomer, CuInS2@ZnS/TiO2 NTs as the basic photoelectrode. With wavelength λ > 240 nm, the photocurrent of MIPPFOA enhanced at higher concentrations of PFOA. With increasing AP concentration, the photocurrents of MIPAP could decline with λ < 271 nm, not change at λ = 270 nm, or increase with λ > 270 nm. As RT concentration increased, the photocurrents of MIPRT could decrease (λ < 431 nm), not change (λ = 431 nm) or increase (λ > 431 nm). The PEC responses depend on the comprehensive interaction of two contrary mechanisms from the template molecules within the MIP membrane. The photocurrent is enhanced by the role of the electron donor for photo-generated holes but attenuated due to the steric hindrance effect and the excitation light intensity loss via absorption or scattering. The apparent molar absorption coefficient of AP and RT within MIP membranes are 9.1-19.4 folds of those measured from dilute solutions. By using a routine UV lamp as the light source, the photocurrents of MIPRT at 254 nm and MIPAP at 365 nm were used to determine RT and AP, with the detection limits of 5.3 and 16 nM, respectively. The interference from the non-specific adsorption of interferents on the surfaces of MIPAP and MIPRT was reduced by one order of magnitude via a differential strategy.

Colchicine protects against the development of experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by at least 1.5-fold enlargement of the infrarenal aorta, a ruptured AAA is life-threatening. Colchicine is a medicine used to treat gout and familial Mediterranean fever, and recently, it was approved to reduce the risk of cardiovascular events in adult patients with established atherosclerotic disease. With an AAA mice model created by treatment with porcine pancreatic elastase (PPE) and ß-aminopropionitrile (BAPN), this work was designed to explore whether colchicine could protect against the development of AAA. Here, we showed that colchicine could limit AAA formation, as evidenced by the decreased total aortic weight per body weight, AAA incidence, maximal abdominal aortic diameter and collagen deposition. We also found that colchicine could prevent the phenotypic switching of vascular smooth muscle cells from a contractile to synthetic state during AAA. In addition, it was demonstrated that colchicine was able to reduce vascular inflammation, oxidative stress, cell pyroptosis and immune cells infiltration to the aortic wall in the AAA mice model. Finally, it was proved that the protective action of colchicine against AAA formation was mainly mediated by preventing immune cells infiltration to the aortic wall. In summary, our findings demonstrated that colchicine could protect against the development of experimental AAA, providing a potential therapeutic strategy for AAA intervention in the clinic.

Intrareticular Charge Transfer Triggered Self-Electrochemiluminescence of Zirconium-Based Metal-Organic Framework Nanoparticles for Potential-Resolved Multiplex Immunoassays with Isolated Coreactants.

In this work, a potential-resolved electrochemiluminescence (ECL) multiplex immunoassay (MIA) was developed using zirconium-based metal-organic framework (MOF) nanoparticles with intense self-ECL as an anodic ECL tag and CdTe nanocrystals (NCs) as a cathodic ECL tag. ECL luminophore 5,5'-(anthracene-9,10-diyl)diisophthalic acid (H4ADIP) and coreactant hexamethylenetetramine (HMT) bound to zirconium nodes in the MOF, giving Zr-ADIP-HMT nanoparticles. Benefiting from the intrareticular charge transfer (ICT) between the oxidized ligands of H4ADIP and HMT via hydrogen bonds, the intense self-ECL from Zr-ADIP-HMT was applied to the potential-resolved ECL MIA without an exogenous anodic coreactant, which can eliminate detrimental effects of multiplex coreactants and anodic ECL emission from CdTe NCs. The ICT within Zr-ADIP-HMT nanoparticles could shorten the electron transport path and reduce the complexity of radical intermediate transport. The ECL intensity from Zr-ADIP-HMT was 18.6-fold that from the mixture of H4ADIP and HMT. In potential-resolved ECL MIA, two lung cancer biomarkers, carcinoembryonic antigen and neuron-specific enolase, were adopted as model analytes, with detection limits of 18 and 5.3 fg·mL-1, respectively. The dual-ligand Zr-ADIP-HMT nanoparticles provide a proof of concept using ICT-based self-ECL luminophores for potential-resolved ECL MIAs with isolated coreactants.

Targeting a novel inducible GPX4 alternative isoform to alleviate ferroptosis and treat metabolic-associated fatty liver disease.

Metabolic-associated fatty liver disease (MAFLD), which is previously known as non-alcoholic fatty liver disease (NAFLD), represents a major health concern worldwide with limited therapy. Here, we provide evidence that ferroptosis, a novel form of regulated cell death characterized by iron-driven lipid peroxidation, was comprehensively activated in liver tissues from MAFLD patients. The canonical-GPX4 (cGPX4), which is the most important negative controller of ferroptosis, is downregulated at protein but not mRNA level. Interestingly, a non-canonical GPX4 transcript-variant is induced (inducible-GPX4, iGPX4) in MAFLD condition. The high fat-fructose/sucrose diet (HFFD) and methionine/choline-deficient diet (MCD)-induced MAFLD pathologies, including hepatocellular ballooning, steatohepatitis and fibrosis, were attenuated and aggravated, respectively, in cGPX4-and iGPX4-knockin mice. cGPX4 and iGPX4 isoforms also displayed opposing effects on oxidative stress and ferroptosis in hepatocytes. Knockdown of iGPX4 by siRNA alleviated lipid stress, ferroptosis and cell injury. Mechanistically, the triggered iGPX4 interacts with cGPX4 to facilitate the transformation of cGPX4 from enzymatic-active monomer to enzymatic-inactive oligomers upon lipid stress, and thus promotes ferroptosis. Co-immunoprecipitation and nano LC-MS/MS analyses confirmed the interaction between iGPX4 and cGPX4. Our results reveal a detrimental role of non-canonical GPX4 isoform in ferroptosis, and indicate selectively targeting iGPX4 may be a promising therapeutic strategy for MAFLD.

Exerkine fibronectin type-III domain-containing protein 5/irisin-enriched extracellular vesicles delay vascular ageing by increasing SIRT6 stability.

AIMS: Exercise confers protection against cardiovascular ageing, but the mechanisms remain largely unknown. This study sought to investigate the role of fibronectin type-III domain-containing protein 5 (FNDC5)/irisin, an exercise-associated hormone, in vascular ageing. Moreover, the existence of FNDC5/irisin in circulating extracellular vesicles (EVs) and their biological functions was explored. METHODS AND RESULTS: FNDC5/irisin was reduced in natural ageing, senescence, and angiotensin II (Ang II)-treated conditions. The deletion of FNDC5 shortened lifespan in mice. Additionally, FNDC5 deficiency aggravated vascular stiffness, senescence, oxidative stress, inflammation, and endothelial dysfunction in 24-month-old naturally aged and Ang II-treated mice. Conversely, treatment of recombinant irisin alleviated Ang II-induced vascular stiffness and senescence in mice and vascular smooth muscle cells. FNDC5 was triggered by exercise, while FNDC5 knockout abrogated exercise-induced protection against Ang II-induced vascular stiffness and senescence. Intriguingly, FNDC5 was detected in human and mouse blood-derived EVs, and exercise-induced FNDC5/irisin-enriched EVs showed potent anti-stiffness and anti-senescence effects in vivo and in vitro. Adeno-associated virus-mediated rescue of FNDC5 specifically in muscle but not liver in FNDC5 knockout mice, promoted the release of FNDC5/irisin-enriched EVs into circulation in response to exercise, which ameliorated vascular stiffness, senescence, and inflammation. Mechanistically, irisin activated DnaJb3/Hsp40 chaperone system to stabilize SIRT6 protein in an Hsp70-dependent manner. Finally, plasma irisin concentrations were positively associated with exercise time but negatively associated with arterial stiffness in a proof-of-concept human study. CONCLUSION: FNDC5/irisin-enriched EVs contribute to exercise-induced protection against vascular ageing. These findings indicate that the exerkine FNDC5/irisin may be a potential target for ageing-related vascular comorbidities.

Activating α7nAChR ameliorates abdominal aortic aneurysm through inhibiting pyroptosis mediated by NLRP3 inflammasome.

Abdominal aortic aneurysm (AAA) is defined as a dilated aorta in diameter at least 1.5 times of a normal aorta. Our previous studies found that activating α7 nicotinic acetylcholine receptor (α7nAChR) had a protective effect on vascular injury. This work was to investigate whether activating α7nAChR could influence AAA formation and explore its mechanisms. AAA models were established by angiotensin II (Ang II) infusion in ApoE-/- mice or in wild type and α7nAChR-/- mice. In vitro mouse aortic smooth muscle (MOVAS) cells were treated with tumor necrosis factor-α (TNF-α). PNU-282987 was chosen to activate α7nAChR. We found that cell pyroptosis effector GSDMD and NLRP3 inflammasome were activated in abdominal aorta, and inflammatory cytokines in serum were elevated in AAA models of ApoE-/- mice. Activating α7nAChR reduced maximal aortic diameters, preserved elastin integrity and decreased inflammatory responses in ApoE-/- mice with Ang II infusion. While α7nAChR-/- mice led to aggravated aortic injury and increased inflammatory cytokines with Ang II infusion when compared with wild type. Moreover, activating α7nAChR inhibited NLRP3/caspase-1/GSDMD pathway in AAA model of ApoE-/- mice, while α7nAChR deficiency promoted this pathway. In vitro, N-acetylcysteine (NAC) inhibited NLRP3 inflammasome activation and NLRP3 knockdown reduced GSDMD expression, in MOVAS cells treated with TNF-α. Furthermore, activating α7nAChR inhibited oxidative stress, reduced NLRP3/GSDMD expression, and decreased cell pyroptosis in MOVAS cells with TNF-α. In conclusion, our study found that activating α7nAChR retarded AAA through inhibiting pyroptosis mediated by NLRP3 inflammasome. These suggested that α7nAChR would be a potential pharmacological target for AAA.

Inhibition of CDK9 attenuates atherosclerosis by inhibiting inflammation and phenotypic switching of vascular smooth muscle cells.

BACKGROUND: Recent studies have demonstrated a key role of vascular smooth muscle cell (VSMC) dysfunction in atherosclerosis. Cyclin-dependent kinases 9 (CDK9), a potential biomarker of atherosclerosis, was significantly increased in coronary artery disease patient serum and played an important role in inflammatory diseases. This study was to explore the pharmacological role of CDK9 inhibition in attenuating atherosclerosis. METHODS: A small-molecule CDK9 inhibitor, LDC000067, was utilized to treat the high fat diet (HFD)-fed ApoE-/- mice and human VSMCs. RESULTS: The results showed that inflammation and phenotypic switching of VSMCs were observed in HFD-induced atherosclerosis in ApoE-/- mice, which were accompanied with increased CDK9 in the serum and atherosclerotic lesions where it colocalized with VSMCs. LDC000067 treatment significantly suppressed HFD-induced inflammation, proliferation and phenotypic switching of VSMCs, resulting in reduced atherosclerosis in the ApoE-/- mice, while had no effect on plasma lipids. Further in vitro studies confirmed that LDC000067 and siRNA-mediated CDK9 knockdown reversed ox-LDL-induced inflammation and phenotypic switching of VSMCs from a contractile phenotype to a synthetic phenotype via inhibiting NF-κB signaling pathway in human VSMCs. CONCLUSION: These results indicate that inhibition of CDK9 may be a novel therapeutic target for the prevention of atherosclerosis.

TLR2 regulates angiotensin II-induced vascular remodeling and EndMT through NF-κB signaling.

Excessive vascular remodeling has been shown in hypertensive patients. In experimental models of hypertensive vascular injury, such as angiotensin II (Ang II) challenged mice, toll like receptor 2 (TLR2) initiates inflammatory responses. More recently, studies have reported atypical endothelial to mesenchymal transition (EndMT) in vascular injuries and inflammatory conditions. Here, we aimed to investigate whether TLR2 mediates Ang II-induced vascular inflammation and initiates EndMT. In a mouse model of angiotensin II-induced hypertension, we show that aortas exhibit increased medial thickening, fibrosis, and features of EndMT. These alterations were not observed in TLR2 knockout mice in response to Ang II. TLR2 silencing in cultured endothelial cells confirmed the essential role of TLR2 in Ang II-induced inflammatory factor induction, and EndMT-associated phenotypic change. Mechanistically, we found Ang II activates nuclear factor-κB signaling, inducing pro-inflammatory cytokine production, and mediates EndMT in both cultured endothelial cells and in mice. These studies illustrate a novel role of TLR2 in regulating Ang II-induced deleterious vascular remodeling through the induction of EndMT. The studies also suggest that TLR2 may be targeted to alleviate hypertension-associated vascular injury.

Identification of RyR2-PBmice and the effects of transposon insertional mutagenesis of the RyR2 gene on cardiac function in mice.

Ryanodine receptor 2 (RyR2) plays an important role in maintaining the normal heart function, and mutantions can lead to arrhythmia, heart failure and other heart diseases. In this study, we successfully identified a piggyBac translocated RyR2 gene heterozygous mouse model (RyR2-PBmice) by tracking red fluorescent protein (RFP) and genotyping PCR. Cardiac function tests showed that there was no significant difference between the RyR2-PBmice and corresponding wild-type mice (WTmice), regardless of whether they were in the basal state or injected with epinephrine and caffeine. However, the sarcoplasmic reticulum Ca2+ content was significantly reduced in the cardiomyocytes of RyR2-PBmice as assessed by measuring caffeine-induced [Ca2+]i transients; the cardiac muscle tissue of RyR2-PBmice displayed significant mitochondrial swelling and focal dissolution of mitochondrial cristae, and the tissue ATP content in the RyR2-PBmice heart was significantly reduced. To further analyze the molecular mechanism behind these changes, we tested the expression levels of related proteins using RT-PCR and Western blot analyses. The mRNA level of RyR2 in RyR2-PBmice cardiac tissue decreased significantly compared with the WTmice, and the protein expression associated with the respiratory chain was also downregulated. These results suggested that the piggyBac transposon inserted into the RyR2 gene substantively affected the structure and function of mitochondria in the mouse cardiomyocytes, leading to disorders of energy metabolism.

Unfound Associated Resonant Model and Its Impact on Response of a Quartz Crystal Microbalance in the Liquid Phase.

Quartz crystal microbalance (QCM) is an important tool to detect in real time the mass change at the nanogram level. However, for a QCM operated in the liquid phase, the Sauerbrey equation is usually disturbed by the changes in liquid properties and the longitudinal wave effect. Herein, we report another unfound associated high-frequency resonance (HFR) model for the QCM, with the intensity 2 orders of magnitude higher than that of the fundamental peak in the liquid phase. The HFR model exhibits obvious impact on the response of QCM in the thickness-shear model (TSM), especially for overtones. The frequency of HFR peak is decreased dramatically with increasing conductivity or permittivity of the liquid phase, resulting in considerable additional frequency shifts in the TSM as baseline drift. Compared to that with a faraway HFR peak, the overlapping of HFR peak to a TSM overtone results in the frequency shifts of ±50-70 kHz with its intensity enhancement by 3 orders of magnitude in the later. The HFR behavior is explained by an equivalent circuit model including leading wire inductance, liquid inductance, and static capacitance of QCM. Taking into account the HFR model, the positive frequency shifts of the QCM at high overtones during the cell adhesion process is understandable. Combining the TSM and HFR is an effective way to improve the stability of QCM and provides more reliable information from the responses of QCM. The HFR may have potential application in chemical and biological sensors.