Prevalence of intracerebral thrombus detected by optical coherence tomography in patients with posterior circulation stroke or transient ischemic attack.

BACKGROUND: The incidence of thrombosis in patients with intracranial atherosclerotic stenosis (ICAS) remains unclear. Optical coherence tomography (OCT) has the potential to explore the vessel wall structure of posterior-circulation ICAS because of its relatively straight anatomical structure compared with that of the anterior cerebral arteries. This study aimed to determine the prevalence and characteristics of thrombosis in the posterior-circulation ICAS using OCT. METHODS: This prospective study was conducted on 135 patients with posterior-circulation arterial stenosis who underwent OCT. All patients were symptomatic and had a severely stenotic lesion (70-99%) in the vetebrobasilar artery. The enrolled patients were classified according to the presence of in situ thrombus as defined by OCT. Clinical data and OCT characteristics were compared. RESULTS: Eighty-two patients diagnosed with posterior-circulation ICAS were enrolled. In situ thrombi were identified in 34 patients. Clinically, patients with in situ thrombus were more prone to cerebral infarctions than transient ischemic attacks. The percentage area of stenosis in the non-thrombus group was significantly lower than that in the thrombus group. The thrombus burden, mean flow area, mean thrombus area, maximum lipid arc, and mean lumen area were significantly different among white, red, and mixed thrombi. CONCLUSIONS: We achieved in vivo vessel wall structural analysis of posterior-circulation ICAS with the largest sample size. We also revealed the true incidence of in situ thrombosis and potential corresponding clinical events of posterior-circulation ICAS for the first time.

Bleeding risk prediction after acute myocardial infarction-integrating cancer data: the updated PRECISE-DAPT cancer score.

BACKGROUND AND AIMS: This study assessed the impact of incorporating cancer as a predictor on performance of the PRECISE-DAPT score. METHODS: A nationally linked cohort of ST-elevation myocardial infarction patients between 1 January 2005 and 31 March 2019 was derived from the UK Myocardial Ischaemia National Audit Project and the UK Hospital Episode Statistics Admitted Patient Care registries. The primary outcome was major bleeding at 1 year. A new modified score was generated by adding cancer as a binary variable to the PRECISE-DAPT score using a Cox regression model and compared its performance to the original PRECISE-DAPT score. RESULTS: A total of 216 709 ST-elevation myocardial infarction patients were included, of which 4569 had cancer. The original score showed moderate accuracy (C-statistic .60), and the modified score showed modestly higher discrimination (C-statistics .64; hazard ratio 1.03, 95% confidence interval 1.03-1.04) even in patients without cancer (C-statistics .63; hazard ratio 1.03, 95% confidence interval 1.03-1.04). The net reclassification index was .07. The bleeding rates of the modified score risk categories (high, moderate, low, and very low bleeding risk) were 6.3%, 3.8%, 2.9%, and 2.2%, respectively. According to the original score, 65.5% of cancer patients were classified as high bleeding risk (HBR) and 21.6% were low or very low bleeding risk. According to the modified score, 94.0% of cancer patients were HBR, 6.0% were moderate bleeding risk, and no cancer patient was classified as low or very low bleeding risk. CONCLUSIONS: Adding cancer to the PRECISE-DAPT score identifies the majority of patients with cancer as HBR and can improve its discrimination ability without undermining its performance in patients without cancer.

Cigarette tar accelerates atherosclerosis progression via RIPK3-dependent necroptosis mediated by endoplasmic reticulum stress in vascular smooth muscle cells.

BACKGROUND: Tar is the main toxic of cigarettes, and its effect on atherosclerosis progression and the underlying mechanisms remain largely unknown. Vascular smooth muscle cells (VSMCs) play a key role in atherogenesis and plaque vulnerability. The present study sought to investigate the mechanism of atherosclerosis progression through tar-induced VSMC necroptosis, a recently described form of necrosis. METHODS: The effect of tar on atherosclerosis progression and VSMC necroptosis was examined in ApoE-/- mice and cultured VSMCs. The role of necroptosis in tar-induced plaque development was evaluated in RIPK3-deletion mice (ApoE-/-RIPK3-/-). The key proteins of necroptosis in carotid plaques of smokers and non-smokers were also examined. Quantitative proteomics of mice aortas was conducted to further investigate the underlying mechanism. Pharmacological approaches were then applied to modulate the expression of targets to verify the regulatory process of tar-induced necroptosis. RESULTS: Tar administration led to increased atherosclerotic plaque area and reduced collagen and VSMCs in ApoE-/- mice. The expression of RIPK1、RIPK3、and MLKL in VSMCs of plaques were all increased in tar-exposed mice and smokers. RIPK3 deletion protected against VSMC loss and plaque progression stimulated by tar. In mechanistic studies, quantitative proteomics analysis of ApoE-/- mice aortas suggested that tar triggered endoplasmic reticulum (ER) stress. PERK-eIF2α-CHOP axis was activated in tar-treated VSMCs and atherosclerotic plaque. Inhibition of ER stress using 4PBA significantly reduced plaque progression and VSMC necroptosis. Further study revealed that ER stress resulted in calcium (Ca2+) release into mitochondria and cytoplasm. Elevated Ca2+ levels lead to mitochondrial dysfunction and excessive reactive oxygen species (ROS) production, which consequently promote RIPK3-dependent necroptosis. In addition, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activated by cytosolic Ca2+ overload binds to RIPK3, accounting for necroptosis. CONCLUSION: The findings revealed that cigarette tar promoted atherosclerosis progression by inducing RIPK3-dependent VSMC necroptosis and identified novel avenues of ER stress and Ca2+ overload.

MCL attenuates atherosclerosis by suppressing macrophage ferroptosis via targeting KEAP1/NRF2 interaction.

BACKGROUND: Micheliolide (MCL), which is the active metabolite of parthenolide, has demonstrated promising clinical application potential. However, the effects and underlying mechanisms of MCL on atherosclerosis are still unclear. METHOD: ApoE-/- mice were fed with high fat diet, with or without MCL oral administration, then the plaque area, lipid deposition and collagen content were determined. In vitro, MCL was used to pretreat macrophages combined by ox-LDL, the levels of ferroptosis related proteins, NRF2 activation, mitochondrial function and oxidative stress were detected. RESULTS: MCL administration significantly attenuated atherosclerotic plaque progress, which characteristics with decreased plaque area, less lipid deposition and increased collagen. Compared with HD group, the level of GPX4 and xCT in atherosclerotic root macrophages were increased in MCL group obviously. In vitro experiment demonstrated that MCL increased GPX4 and xCT level, improved mitochondrial function, attenuated oxidative stress and inhibited lipid peroxidation to suppress macrophage ferroptosis induced with ox-LDL. Moreover, MCL inhibited KEAP1/NRF2 complex formation and enhanced NRF2 nucleus translocation, while the protective effect of MCL on macrophage ferroptosis was abolished by NRF2 inhibition. Additionally, molecular docking suggests that MCL may bind to the Arg483 site of KEAP1, which also contributes to KEAP1/NRF2 binding. Furthermore, Transfection Arg483 (KEAP1-R483S) mutant plasmid can abrogate the anti-ferroptosis and anti-oxidative effects of MC in macrophages. KEAP1-R483S mutation also limited the protective effect of MCL on atherosclerosis progress and macrophage ferroptosis in ApoE-/- mice. CONCLUSION: MCL suppressed atherosclerosis by inhibiting macrophage ferroptosis via activating NRF2 pathway, the related mechanism is through binding to the Arg483 site of KEAP1 competitively.

Apigenin inhibits macrophage pyroptosis through regulation of oxidative stress and the NF-κB pathway and ameliorates atherosclerosis.

Pyroptosis plays an important role in inflammatory diseases such as viral hepatitis and atherosclerosis. Apigenin exhibits various bioactivities, particularly anti-inflammation, but its effect on pyroptosis remains unclear. The aim of this study is to investigate the effect of apigenin on pyroptosis and explore its potential against inflammatory diseases. THP-1 macrophages treated by lipopolysaccharides/adenosine 5'-triphosphate were used as the in vitro pyroptosis model. Western blot was used to detect the expression of NLRP3 inflammasome components and key regulators. Immunofluorescence was used to observe ROS production and intracellular location of p65. The potential of apigenin against inflammatory diseases was evaluated using atherosclerotic mice. Plaque progression was observed by pathological staining. Immunofluorescence was used to observe the expression of NLRP3 inflammasome components in plaques. The results showed that apigenin inhibited NLRP3 inflammasome activation. Apigenin reduced ROS overproduction and inhibited p65 nuclear translocation. Additionally, apigenin decreased the expression of NLRP3 inflammasome components in the plaque. Plaque progression was inhibited by apigenin. In conclusion, apigenin exhibited a preventive effect on macrophage pyroptosis by reducing oxidative stress and inhibiting the NF-κB pathway. Apigenin may alleviate atherosclerosis at least partially by inhibiting macrophage pyroptosis. These findings suggest apigenin to be a promising therapeutic agent for inflammatory diseases.

Estrogen deficiency accelerates postmenopausal atherosclerosis by inducing endothelial cell ferroptosis through inhibiting NRF2/GPX4 pathway.

Oxidative stress and lipid metabolism disorder caused by estrogen deficiency are regarded as the main causes of postmenopausal atherosclerosis, but the underlying mechanisms remain still unclear. In this study, ovariectomized (OVX) female ApoE-/- mice fed with high-fat diet were used to imitate postmenopausal atherosclerosis. The atherosclerosis progression was significantly accelerated in OVX mice, accompanied by the upregulation of ferroptosis indicators, including increased lipid peroxidation and iron deposition in the plaque and the plasma. While both estradiol (E2) and ferroptosis inhibitor ferrostatin-1 alleviated atherosclerosis in OVX mice, with the inhibition of lipid peroxidation and iron deposition, as well as the upregulation of xCT and GPX4, especially in endothelial cells. We further investigated the effects of E2 on ferroptosis in endothelial cells induced by oxidized-low-density lipoprotein or ferroptosis inducer Erastin. It was found that E2 exhibited anti-ferroptosis effect through antioxidative functions, including improving mitochondrial dysfunction and upregulating GPX4 expression. Mechanistically, NRF2 inhibition attenuated the effect of E2 against ferroptosis as well as the upregulation of GPX4. Our findings revealed that endothelial cell ferroptosis played a pivotal role in postmenopausal atherosclerosis progression, and the NRF2/GPX4 pathway activation contributed to the protection of E2 against endothelial cell ferroptosis.

Cigarette tar mediates macrophage ferroptosis in atherosclerosis through the hepcidin/FPN/SLC7A11 signaling pathway.

Despite the known promotional effects of cigarette smoking on progression of atherosclerosis (AS), tar as the most dominant toxic component in cigarette smoking has been little studied. Understanding the potential role and mechanisms of tar in AS may be a prerequisite for future reductions in cardiovascular morbidity and mortality. Male ApoE-/- mice were fed with high-fat diet and injected intraperitoneally with cigarette tar (40 mg/kg/day) for 16 weeks. The results showed that cigarette tar significantly promoted the formation of lipid-rich plaques with larger necrotic cores and less fibrous, and caused severe iron overload and lipid peroxidation in AS lesions. Moreover, tar significantly upregulated the expression of hepcidin and downregulated FPN and SLC7A11 of macrophages in AS plaques. Ferroptosis inhibitor (FER-1 and DFO) treatment, hepcidin-knockdown or SLC7A11-overexpression reversed above changes, thereby delaying the progression of atherosclerosis. In vitro, the use of FER-1, DFO, si-hepcidin, and ov-SLC7A11 increased cell viability and inhibited iron accumulation, lipid peroxidation and GSH depletion in tar treated macrophages. These interventions also inhibited the tar induced upregulation of hepcidin, and increased the expression of FPN, SLC7A11, and GPX4. Furthermore, NF-κB inhibitor reversed the regulatory effect of tar on hepcidin/FPN/SLC7A11 axis, and then inhibiting macrophage ferroptosis. These findings indicated that cigarette tar promotes atherosclerosis progression by inducing macrophage ferroptosis via NF-κB-activated hepcidin/FPN/SLC7A11 pathway.

A Nrf2-OSGIN1&2-HSP70 axis mediates cigarette smoke-induced endothelial detachment: implications for plaque erosion.

AIMS: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion. METHODS AND RESULTS: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture. CONCLUSION: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

LncRNA MEG3 attenuates the malignancy of retinoblastoma cells through inactivating PI3K /Akt/mTOR signaling pathway.

Retinoblastoma (RB) is the most common neoplasm found in the eye of children. There are increasing interests to develop targeted gene therapy for this disease. This study was performed to investigate the impact of long non-coding RNA (lncRNA) MEG3 on the biological features of RB cells. Vector overexpressing MEG3 was constructed and introduced into two RB cell lines. Transfected RB cells were assessed for proliferation, apoptosis, migration ability, expression levels of important genes in the PI3K/Akt/mTOR signaling pathway using qRT-PCR and Western blot analysis. Xenograft mouse models were constructed to determine the tumorigenicity of RB cells overexpressing MEG3. MEG3 mRNA level was significantly lower in RB cells than in non-cancer cells (p < 0.01). Overexpressing MEG3 resulted in significant reduction in cell proliferation (p < 0.05), migration (p < 0.01) and significant increase in apoptosis (p < 0.01). After overexpressing MEG3, p-PI3K, p-Akt and p-mTOR levels were significantly downregulated (p < 0.01). Furthermore, in the xenograft model, RB cells overexpressing MEG3 generated significantly smaller tumors as compared to RB cells that did not overexpress MEG3 (p < 0.05). Our data suggest that MEG3 increases apoptosis and reduces tumorigenicity of RB cells through inactivating the PI3K/Akt/mTOR pathway. Therefore, MEG3 could be further investigated as a potential new therapeutic agent and target for RB therapy.

A novel anti-atherosclerotic mechanism of quercetin: Competitive binding to KEAP1 via Arg483 to inhibit macrophage pyroptosis.

Natural antioxidants represented by quercetin have been documented to be effective against atherosclerosis. However, the related mechanisms remain largely unclear. In this study, we identified a novel anti-atherosclerotic mechanism of quercetin inhibiting macrophage pyroptosis by activating NRF2 through binding to the Arg483 site of KEAP1 competitively. In ApoE-/- mice fed with high fat diet, quercetin administration attenuated atherosclerosis progression by reducing oxidative stress level and suppressing macrophage pyroptosis. At the cellular level, quercetin suppressed THP-1 macrophage pyroptosis induced by ox-LDL, demonstrated by inhibiting NLRP3 inflammasome activation and reducing ROS level, while these effects were reversed by the specific NRF2 inhibitor (ML385). Mechanistically, quercetin promoted NRF2 to dissociate from KEAP1, enhanced NRF2 nuclear translocation as well as transcription of downstream antioxidant protein. Molecular docking results suggested that quercetin could bind with KEAP1 at Arg415 and Arg483. In order to verify the binding sites, KEAP1 mutated at Arg415 and Arg483 to Ser (R415S and R483S) was transfected into THP-1 macrophages, and the anti-pyroptotic effect of quercetin was abrogated by Arg483 mutation, but not Arg415 mutation. Furthermore, after administration of adeno associated viral vector (AAV) with AAV-KEAP1-R483S, the anti-atherosclerotic effects of quercetin were almost abolished in ApoE-/- mice. These findings proved quercetins suppressed macrophage pyroptosis by targeting KEAP1/NRF2 interaction, and provided reliable data on the underlying mechanism of natural antioxidants to protect against atherosclerosis.

lncRNA MEG3 Inhibits the Proliferation and Growth of Glioma Cells by Downregulating Bcl-xL in the PI3K/Akt/NF-κB Signal Pathway.

This study was conducted to investigate the impact and mechanisms of lncRNA MEG3 on glioma cells. lncRNA MEG3 was lowly expressed in glioma cells as compared to noncancer cells. Overexpression of MEG3 significantly downregulated the expression of Bcl-xL, slightly upregulated the expression of NF-κB p65 and IκBα, and reduced the proliferation of glioma cells with increased apoptosis and the migration and invasion ability. Subsequently, glioma cells overexpressing MEG3 had less tumorgenicity in xenograft mouse models. It is likely that MEG3 induces apoptosis in glioma cells via downregulating the Bcl-xL gene in the PI3K/Akt/NF-κB signal pathway to reduce the development of glioma.

Biodegradable electrospun nanofibrous platform integrating antiplatelet therapy-chemotherapy for preventing postoperative tumor recurrence and metastasis.

The perioperative trauma-related platelet recruitment and activation severely affect tumor recurrence and metastasis. Therefore, efficiently killing residual tumor cells and simultaneously inhibiting platelet activation to block platelet-cancer cell interaction might be a promising strategy to prevent postoperative tumor recurrence and metastasis. Methods: Biodegradable PLGA electrospun nanofibrous films co-delivering doxorubicin-loaded tumor repopulating cell-derived microparticles (DOX-MPs) and aspirin (ASA) were developed as the implant materials (DOX-MPs/ASA@NF) for postoperative in-situ treatment. The characterization, cytotoxicity against tumor cells, inhibition in platelet activation-triggered proliferation, migration and metastasis of tumor cells and in vivo anti-recurrence and anti-metastasis activity induced by DOX-MPs/ASA@NF were systematically evaluated. Results: PLGA nanofibrous films facilitate the enhanced distribution of DOX-MPs as well as DOX-MPs and ASA release in a time-programmed manner within the tumor resection cavity. The released DOX-MPs efficiently kill the residual tumor cells, while ASA decreases platelet activation and inhibits platelet-promoted proliferation, migration and metastasis of tumor cells, resulting in the remarkable inhibition of postoperative tumor recurrence and metastasis. Conclusions: DOX-MPs/ASA@NF may be a promising candidate to prevent the recurrence and metastasis of resectable tumors.

Prevalence and prognostic significance of DNMT3A- and TET2- clonal haematopoiesis-driver mutations in patients presenting with ST-segment elevation myocardial infarction.

BACKGROUND: Clonal haematopoiesis driven by mutations in DNMT3A or TET2 has recently been identified as a new risk factor for cardiovascular disease. Experimental studies suggest that these mutations may enhance inflammation which accelerates the disease progression. We aim to investigate the prevalence of mutations in DNMT3A and TET2 and their association with prognosis of patients with ST-segment elevation myocardial infarction (STEMI). METHODS: Targeted deep sequencing for DNMT3A and TET2 and inflammatory cytokines (IL-1ß, IL-6, TNF-α, INF-γ) were analyzed in 485 patients with STEMI. Major adverse cardiac events (MACE) was a composite of death, myocardial infarction, stroke, or hospitalization due to heart failure. FINDINGS: Patients carrying DNMT3A- or TET2-CH-driver mutations with a variant allele frequency (VAF) ≥2% were found in 12.4% (60 of 485) of STEMI patients and experienced an increased incidence of the death (30.9% vs 15.5%, P = 0.001) and MACE (44.5% vs 21.8%, P < 0.001) compared to those who did not, during a median follow up of 3.0 (interquartile range: 2.4-3.4) years. After adjusting for confounders, mutation remained an independent predictor of death (HR = 1.967, 95% CI 1.103-3.507, P = 0.022) and MACE (HR = 1.833, 95% CI 1.154-2.912, P = 0.010). Concentrations of plasma IL-1ß (P = 0.010) and IL-6 (P = 0.011) were significantly elevated in DNMT3A/TET2 VAF≥2% group. INTERPRETATION: DNMT3A- or TET2-CH-driver mutations with a VAF≥2% were observed in over 10% STEMI patients, and were significantly associated with poorer prognosis, which might be explained by higher levels of inflammatory cytokines in mutations carriers. FUNDING: National Natural Science Foundation of China; National Key R&D Program of China.

Conformation-reconstructed multivalent antibody mimic for amplified mitigation of human islet amyloid polypeptide amyloidogenesis.

The misfolding and aggregation of human islet amyloid polypeptide (IAPP) into ß-sheet-enriched amyloid fibrils is linked to type 2 diabetes. Antibodies are potent inhibitors of IAPP amyloidogenesis, but their preparation is usually complicated and expensive. Here we have created a multivalent antibody mimic SPEPS@Au through conformational engineering of the complementary-determining regions (CDRs) of antibodies on gold nanoparticles (AuNPs). By immobilizing both terminals of an IAPP-recognizing CDR loop (PEP) on the surface of AuNPs, the active conformation of PEP can simply recur on the gold-based antibody mimic, significantly enhancing the binding affinity between PEP and IAPP. SPEPS@Au mitigated amyloidogenesis of IAPP at low sub-stoichiometric concentrations, even after IAPP started aggregating, and dramatically reduced the amyloidogenesis-induced toxicity and ROS production both in vitro and in vivo. The conformation-reconstructed multivalent antibody mimic not only renders a facile strategy to approach potent amyloidogenesis inhibitors, but also provides new perspectives to exploit NP-based substitutes for antibodies in various applications.

Comprehensive Assessment of Coronary Calcification in Intravascular OCT Using a Spatial-Temporal Encoder-Decoder Network.

Coronary calcification is a strong indicator of coronary artery disease and a key determinant of the outcome of percutaneous coronary intervention. We propose a fully automated method to segment and quantify coronary calcification in intravascular OCT (IVOCT) images based on convolutional neural networks (CNN). All possible calcified plaques were segmented from IVOCT pullbacks using a spatial-temporal encoder-decoder network by exploiting the 3D continuity information of the plaques, which were then screened and classified by a DenseNet network to reduce false positives. A novel data augmentation method based on the IVOCT image acquisition pattern was also proposed to improve the performance and robustness of the segmentation. Clinically relevant metrics including calcification area, depth, angle, thickness, volume, and stent-deployment calcification score, were automatically computed. 13844 IVOCT images with 2627 calcification slices from 45 clinical OCT pullbacks were collected and used to train and test the model. The proposed method performed significantly better than existing state-of-the-art 2D and 3D CNN methods. The data augmentation method improved the Dice similarity coefficient for calcification segmentation from 0.615±0.332 to 0.756±0.222, reaching human-level inter-observer agreement. Our proposed region-based classifier improved image-level calcification classification precision and F1-score from 0.725±0.071 and 0.791±0.041 to 0.964±0.002 and 0.883±0.008, respectively. Bland-Altman analysis showed close agreement between manual and automatic calcification measurements. Our proposed method is valuable for automated assessment of coronary calcification lesions and in-procedure planning of stent deployment.

The protective effect of quercetin on macrophage pyroptosis via TLR2/Myd88/NF-κB and ROS/AMPK pathway.

AIMS: Pyroptosis is a pro-inflammatory form of programmed cell death, which plays a vital role in the development of inflammatory diseases. As a natural flavonoid, quercetin has been shown to possess anti-inflammatory activity, but its effects on macrophage pyroptosis is still unclear. Therefore, this study aims to investigate the effects of quercetin on macrophage pyroptosis and the underlying mechanism. MATERIAL AND METHODS: LPS/ATP treatment was used to induce THP-1 macrophage pyroptosis. Cell counting kit-8 (CCK-8) assay was used to evaluate cell viability. Scanning electron microscope (SEM) was used to detect cell morphology. Hoechst/propidium iodide (PI) staining and lactate dehydrogenase (LDH) assay were performed to evaluate the cell membrane integrity. The expression of key components and effectors of nod-like receptors3 (NLRP3) inflammasome were examined by real-time PCR and western blot. Immunofluorescence staining was used to detect reactive oxygen species (ROS) level and P65 nuclear translocation. KEY FINDINGS: Our results showed that quercetin prevented THP-1 macrophage pyroptosis by reducing the expression of NLRP3 and cleaved-caspase1, as well as IL-1ß and N-GSDMD in a concentration dependent manner. Quercetin suppressed NLRP3 inflammasome activation by inhibiting ROS overproduction. Moreover, quercetin inhibited the phosphorylation of P65 and its translocation from cytoplasm into nuclear. In addition, we found that quercetin suppressed the increase of TLR2/Myd88 and p-AMPK induced by LPS/ATP, while both TLR2 and AMPK agonist weakened the inhibitory effect of quercetin on the activity of NLRP3 inflammasome and alleviated the protective effect on macrophages pyroptosis. SIGNIFICANCE: Quercetin possesses a protective effect on macrophages pyroptosis via TLR2/Myd88/NF-κB and ROS/AMPK pathway.

Hierarchical Vitalization of Oligotyrosine in Mitigating Islet Amyloid Polypeptide Amyloidogenesis through Multivalent Macromolecules with Conformation-Restrained Nanobody Ligands.

The development of inhibitors that can effectively mitigate the amyloidogenesis of human islet amyloid polypeptide (hIAPP), which is linked to type II diabetes, remains a great challenge. Oligotyrosines are intriguing candidates in that they can block the hIAPP aggregation through multiplex phenol-hIAPP interactions. However, oligotyrosines containing too many tyrosine units (larger than three) may fail to inhibit amyloidogenesis due to their increased hydrophobicity and strong self-aggregation propensity. In this work, we developed a strategy to hierarchically vitalize oligotyrosines in mitigating hIAPP amyloidogenesis. Tetratyrosine YYYY (4Y) was grafted into the third complementary-determining region (CDR3) of a parent nanobody to construct a sequence-programmed nanobody N4Y, in which the conformation of the grafted 4Y fragment was constrained for a significantly enhanced binding affinity with hIAPP. We next conjugated N4Y to a polymer to approach a secondary vitalization of 4Y through a multivalent effect. The in vitro and in vivo experiments validated that the resulting PDN4Y could completely inhibit the hIAPP amyloidogenesis at low stoichiometric concentrations and effectively suppress the generation of toxic reactive oxygen species and alleviate amyloidogenesis-mediated damage to INS-1 cells and zebrafish (Danio rerio) embryos. The hierarchical vitalization of 4Y via a synergistic conformation restraint and multivalent effect represents a strategic prototype of boosting the efficacy of peptide-based amyloidogenesis inhibitors, especially those with a high hydrophobicity and strong aggregation tendency, which holds great promise for future translational studies.

Sfxn1 is essential for erythrocyte maturation via facilitating hemoglobin production in zebrafish.

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one­carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.