Copper(II)-Based Nano-Regulator Correlates Cuproptosis Burst and Sequential Immunogenic Cell Death for Synergistic Cancer Immunotherapy.

Immunogenic cell death (ICD) of tumor cells serves as a crucial initial signal in the activation of anti-tumor immune responses, holding marked promise in the field of tumor immunotherapy. However, low immunogenicity tumors pose challenges in achieving complete induction of ICD, thereby limiting the response rates of immunotherapy in clinical patients. The emergence of cuproptosis as a new form of regulated cell death has presented a promising strategy for enhanced immunotherapy of low immunogenic tumors. To trigger cuproptosis, copper-ionophore elesclomol (ES) had to be employed for the copper-transporting-mediated process. Herein, we proposed a copper(II)-based metal-organic framework nanoplatform (Cu-MOF) to facilitate a cooperative delivery of encapsulated ES and copper (ES-Cu-MOF) to induce cuproptosis burst and enhance ICD of fibrosarcoma. Our results showed that the ES-Cu-MOF nano-regulator could effectively release Cu2+ and ES in response to the intracellular environment, resulting in elevated mitochondrial ROS generation and initiated cuproptosis of tumor cells. Furthermore, sequential ICDs were significantly triggered via the ES-Cu-MOF nano-regulator to activate the anti-tumor immune response. The results of tumor inhibition experiment indicated that the nano-regulator of ES-Cu-MOF obviously accumulated in the tumor site, inducing ICD for dendritic cell activation. This enabled an increased infiltration of cytotoxic CD8+ T cells and consequently enhanced antitumor immune responses for successfully suppressing fibrosarcoma growth. Thus, the copper(II)-based metal-organic framework nano-regulator offered a promising approach for inducing cuproptosis and cuproptosis-stimulated ICD for cancer immunotherapy.

Isorhamnetin alleviates ferroptosis-mediated colitis by activating the NRF2/HO-1 pathway and chelating iron.

Ferroptosis of intestinal epithelial cells (IECs) had been identified as a key factor in the development of ulcerative colitis (UC). Therefore, targeted inhibition of ferroptosis may provide a new strategy for the treatment of UC. Isorhamnetin (ISO) was an O-methylated flavonol with therapeutic effects on a variety of diseases, such as cardiovascular disease, neurological disorders and tumors. However, the role and mechanism of ISO in ferroptosis and associated colitis were rarely investigated. In this study, we demonstrated that ISO could effectively alleviate intestinal inflammation by inhibiting ferroptosis of IECs in DSS-induced mice. Moreover, our results shown that ISO acted as a potent and common ferroptosis inhibitor in multiple human and murine cell lines. Mechanistically, ISO inhibited ferroptosis independent of its previously reported targets MEK1 and PI3K, but alleviated oxidative stress by targeting and activating NRF2. Furthermore, ISO could also directly chelate iron to hinder ferroptosis. In conclusion, our study indicated that ISO as a novel potential ferroptosis inhibitor, providing a promising therapeutic strategy for ferroptosis-related colitis.

Site-Specific Analysis of Core and Antenna Fucosylation on Serum Glycoproteins.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N-glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N-glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N-glycans, 37% by CF N-glycans, and 13% by dual-fucosylated N-glycans. Interestingly, CF and AF N-glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N-glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N-glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.

Identification of a targeted ACSL4 inhibitor to treat ferroptosis-related diseases.

Ferroptosis is a form of iron-dependent, lipid peroxidation-driven regulatory cell death that has been implicated in the pathogenesis of multiple diseases, including organ injury, ischemia/reperfusion, and neurodegenerative diseases. However, inhibitors that directly and specifically target ferroptosis are not yet available. Here, we identify the compound AS-252424 (AS) as a potent ferroptosis inhibitor through kinase inhibitor library screening. Our results show that AS effectively inhibits lipid peroxidation and ferroptosis in both human and mouse cells. Mechanistically, AS directly binds to the glutamine 464 of ACSL4 to inhibit its enzymatic activity, resulting in the suppression of lipid peroxidation and ferroptosis. By using nanoparticle-based delivery systems, treatment with AS-loaded nanoparticles effectively alleviate ferroptosis-mediated organ injury in mouse models, including kidney ischemia/reperfusion injury and acute liver injury (ALI). Thus, our results identify that AS is a specific and targeted inhibitor of ACSL4 with remarkable antiferroptosis function, providing a potential therapeutic for ferroptosis-related diseases.

Entrectinib inhibits NLRP3 inflammasome and inflammatory diseases by directly targeting NEK7.

Excessive inflammation caused by abnormal activation of the NLRP3 inflammasome contributes to the pathogenesis of multiple human diseases, but clinical drugs targeting the NLRP3 inflammasome are still not available. In this study, we identify entrectinib (ENB), a US Food and Drug Administration (FDA)-approved anti-cancer agent, as a target inhibitor of the NLRP3 inflammasome to treat related diseases. ENB specifically blocks NLRP3 without affecting activation of other inflammasomes. Furthermore, we demonstrate that ENB directly binds to arginine 121 (R121) of NEK7 and blocks the interaction between NEK7 and NLRP3, thereby inhibiting inflammasome assembly and activation. In vivo studies show that ENB has a significant ameliorative effect on mouse models of NLRP3 inflammasome-related diseases, including lipopolysaccharide (LPS)-induced systemic inflammation, monosodium urate (MSU)-induced peritonitis, and high-fat diet (HFD)-induced type 2 diabetes (T2D). These data show that ENB is a targeted inhibitor of NEK7 with strong anti-NLRP3 inflammasome activity, making it a potential candidate drug for the treatment of inflammasome-related diseases.

Protocol for in vivo and in vitro activation of NLRP3 inflammasome in mice using monosodium urate.

Although intraperitoneal injection of monosodium urate (MSU) is an effective model for studying peritonitis, its establishment remains challenging. Here, we present a protocol for using MSU to activate the NLRP3 inflammasome in bone-marrow-derived macrophages (BMDMs) and to induce peritonitis in mice. We describe steps for isolating and culturing BMDMs, preparing MSU crystals, and activating the NLRP3 inflammasome using western blot and ELISA. We then detail procedures for inducing peritonitis and testing for relevant indicators using flow cytometry and ELISA. For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1.

Role of pyroptosis in the pathogenesis and treatment of diseases.

Programmed cell death (PCD) is regarded as a pathological form of cell death with an intracellular program mediated, which plays a pivotal role in maintaining homeostasis and embryonic development. Pyroptosis is a new paradigm of PCD, which has received increasing attention due to its close association with immunity and disease. Pyroptosis is a form of inflammatory cell death mediated by gasdermin that promotes the release of proinflammatory cytokines and contents induced by inflammasome activation. Recently, increasing evidence in studies shows that pyroptosis has a crucial role in inflammatory conditions like cardiovascular diseases (CVDs), cancer, neurological diseases (NDs), and metabolic diseases (MDs), suggesting that targeting cell death is a potential intervention for the treatment of these inflammatory diseases. Based on this, the review aims to identify the molecular mechanisms and signaling pathways related to pyroptosis activation and summarizes the current insights into the complicated relationship between pyroptosis and multiple human inflammatory diseases (CVDs, cancer, NDs, and MDs). We also discuss a promising novel strategy and method for treating these inflammatory diseases by targeting pyroptosis and focus on the pyroptosis pathway application in clinics.

Serum biomarker panel for disease severity and prognosis in patients with COVID-19.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has become a worldwide emergency and has had a severe impact on human health. Inflammatory factors have the potential to either enhance the efficiency of host immune responses or damage the host organs with immune overreaction in COVID-19. Therefore, there is an urgent need to investigate the functions of inflammatory factors and serum markers that participate in disease progression. METHODS: In total, 54 COVID-19 patients were enrolled in this study. Disease severity was evaluated by clinical evaluation, laboratory tests, and computed tomography (CT) scans. Data were collected at: admission, 3-5 days after admission, when severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection became negative, and composite endpoint. RESULTS: We found that the positive rate in sputum was three times higher than that in throat swabs. Higher levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer (D-D), interleukin-6 (IL-6) and neutrophil-to-lymphocyte ratio (NLR) or lower lymphocyte counts suggested more severe disease, and the levels of cytokines and serum markers were intrinsically correlated with disease progression. When SARS-CoV-2 RNA detection became negative, the receiver operating characteristic (ROC) curve demonstrated that LDH had the highest sensitivity independently, and four indicators (NLR, CRP, LDH, and D-D) when combined had the highest sensitivity in distinguishing critically ill patients from mild ones. CONCLUSIONS: Monitoring dynamic changes in NLR, CRP, LDH, IL-6, and D-D levels, combined with CT imaging and viral RNA detection in sputum, could aid in severity evaluation and prognosis prediction and facilitate COVID-19 treatment.

Application of StrucGP in medical immunology: site-specific N-glycoproteomic analysis of macrophages.

The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals. Meanwhile, the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions. In this work, we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis. The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry (MS)-based glycoproteomic approaches, followed by the large-scale mapping of site-specific glycan structures via StrucGP. Results revealed that bisected GlcNAc, core fucosylated, and sialylated glycans (e.g., HexNAc4Hex5Fuc1Neu5Ac1, N4H5F1S1) were increased in M1 and M2 macrophages, especially in the latter. The findings indicated that these structures may be closely related to macrophage polarization. In addition, a high level of glycosylated PD-L1 was observed in M1 macrophages, and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1, and Asn-200 contained Lewis epitopes. The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.

Recognition of Core-Fucosylated Glycopeptides Based on the Y1+Fuc/Y1 Ratio in Low-Energy HCD Spectra.

Accurate identification of core fucosylation on N-glycopeptides remains challenging due to fucose migration during mass spectrometry analysis. Here, we introduce a simple and straightforward method for core-fucosylated glycopeptide recognition based on the relative intensities of Y1+Fuc ions compared with their corresponding Y1 ions (labeled as Y1+Fuc/Y1 or simply Y1F/Y1 ratio > 0.1) in low-energy HCD-based spectra. The method was first developed by systematically evaluating the influence of fucose migration on the Y1F ion from antenna fucoses based on the distribution of the Y1F/Y1 ratios in the MS/MS spectra of antenna-fucosylated glycopeptides from Fut8-/- mouse brain. The feasibility of the method was then confirmed by using two standard glycoproteins, comparison with glycopeptides in Fut8+/+ mouse brain with/without in silico core-fucosylation removal, and Y1F/Y1 ratio alterations under a lower HCD energy. This method will be applicable to the manual interpretation and software-based high-throughput analysis of core-fucosylated glycopeptides.

Precision N-glycoproteomics reveals elevated LacdiNAc as a novel signature of intrahepatic cholangiocarcinoma.

Primary liver cancer, mainly comprising hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), remains a major global health problem. Although ICC is clinically different from HCC, their molecular differences are still largely unclear. In this study, precision N-glycoproteomic analysis was performed on both ICC and HCC tumors as well as paracancer tissues to investigate their aberrant site-specific N-glycosylation. By using our newly developed glycoproteomic methods and novel algorithm, termed 'StrucGP', a total of 486 N-glycan structures attached on 1235 glycosites were identified from 894 glycoproteins in ICC and HCC tumors. Notably, glycans with uncommon LacdiNAc (GalNAcß1-4GlcNAc) structures were distinguished from their isomeric glycans. In addition to several bi-antennary and/or bisecting glycans that were commonly elevated in ICC and HCC, a number of LacdiNAc-containing, tri-antennary, and core-fucosylated glycans were uniquely increased in ICC. More interestingly, almost all LacdiNAc-containing N-glycopeptides were enhanced in ICC tumor but not in HCC tumor, and this phenomenon was further confirmed by lectin histochemistry and the high expression of ß1-4 GalNAc transferases in ICC at both mRNA and protein expression levels. The novel N-glycan alterations uniquely detected in ICC provide a valuable resource for future studies regarding to the discovery of ICC diagnostic biomarkers, therapeutic targets, and mechanism investigations.

Site-specific glycoproteomic analysis revealing increased core-fucosylation on FOLR1 enhances folate uptake capacity of HCC cells to promote EMT.

Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous. Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-ß1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches. Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-ß1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-ß1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells. Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-ß1-induced EMT of HCC cells: HGF or TGF-ß1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.

Antidiabetic effects of water-soluble Korean pine nut protein on type 2 diabetic mice.

Korean pine nut protein (PNP) has a variety of biological activities, which are good for human health, but its ability to preventing diabetes has not been reported. This study evaluated the effects of water-soluble proteins of Korean pine nut obtained from a dilute alkali extract on carbohydrate metabolism of type 2 diabetic mice on a model of diabetes induced using a high fat diet combined with streptozotocin. The results showed that the hypoglycemic effect of PNP at a middle dose was the most significant, which was 38.7% lower than that of control. The extract significantly improved the oral glucose tolerance and liver indexes, increased the activity of the carbohydrate metabolism enzymes, and regulated the expression of the function of key genes for carbohydrate metabolism. It had a positive effect on both insulin resistance and glycolytic/gluconeogenesis signaling. In conclusion, PNP can regulate fasting blood glucose, improve insulin resistance, correct the glucose metabolism disorder in diabetic mice, and have a positive regulatory role. As the functional food, it has the potential to be beneficial in the treatment of type 2 diabetes mellitus as a new hypoglycemic functional food.

The antitumor effect of folic acid conjugated-Auricularia auricular polysaccharide-cisplatin complex on cervical carcinoma cells in nude mice.

A tumor-targeted, folic acid (FA) conjugated-Auricularia auricular polysaccharide (AAP) -cis-diaminedichloroplatinum (CDDP) complex (FA-AAP-CDDP) was used for cervical carcinoma chemotherapy. The drug delivery system was able to enhance the antitumor potency of CDDP, and to reduce the toxic side effects of CDDP. The kidney of mice treated by FA-AAP-CDDP complex had higher superoxide dismutase, catalase, and glutathione peroxidase activities, and lower malondialdehyde. FA-AAP-CDDP complex could induce more interleukin-2, interleukin-4, and interferon-γ in mice. In addition, the FA-AAP-CDDP complex significantly promoted the expression of Bax and caspase-3 protein, but inhibited the expression of Bcl-2 protein, which activated the mitochondrial apoptotic pathway of tumor cells in nude mice. Moreover, the FA-AAP-CDDP complex had a higher intratumoral accumulation, was lower in the kidneys. This study may provide a new direction for folate receptor targeted polymers to improve anti-tumor activity, but reduce side effects of CDDP.

Effect of polyamines on the grain filling of wheat under drought stress.

Drought inhibits wheat grain filling. Polyamines (PAs) are closely associated with plant resistance due to drought and grain filling of cereals. However, little is known about the effect of PAs on the grain filling of wheat under drought stress. This study investigated whether and how PAs are involved in regulating wheat grain filling under drought stress. Two wheat genotypes differing in drought resistance were used, and endogenous PA levels were measured during grain filling under different water treatments. Additionally, external PAs were used, and the variation of hormone levels in grains was measured during grain filling under drought stress. The results indicated that spermidine (Spd) and spermine (Spm) relieve the inhibition caused by drought stress, and putrescine (Put) has the opposite effect. The higher activities of S-adenosylmethionine decarboxylase and Spd synthase in grains promotes the synthetic route from Put to Spd and Spm and notably increases the free Spd and Spm concentrations in grains, which promotes grain filling and drought resistance in wheat. The effect of PA on the grain filling of wheat under drought stress was closely related to the endogenous ethylene (ETH), zeatin (Z) + zeatin riboside (ZR) and abscisic acid (ABA). Spd and Spm significantly increased the Z + ZR and ABA concentrations and decreased the ETH evolution rate in grains, which promoted wheat grain filling under drought. Put significantly increased the ETH evolution rate, which led to excessive ABA accumulation in grains, subsequently aggravating the inhibition of drought on wheat grain filling. This means that the interaction of hormones, rather than the action of a single hormone, was involved in the regulation of wheat grain filling under drought.

Effect of plastic film mulching on the grain filling and hormonal changes of maize under different irrigation conditions.

Plastic film mulching (PM) is widely utilized for maize production in China. However, the effect of PM on the grain yield of crops has not been established, and the biochemical mechanism underlying the increase or decrease in grain yield under PM is not yet understood. Grain filling markedly affects the grain yield. The objective of this study was to investigate the effects of PM on maize grain filling under different irrigation levels and the relationship of such effects with hormonal changes. In the present study, PM was compared with traditional nonmulching management (TN) under 220 mm, 270 mm and 320 mm irrigation amount, and the grain filling characters of the grains located in various parts of the ear and the hormonal changes in the grains were measured. The results indicated that at 220 mm irrigation, PM significantly increased the grain filling rate of the middle and basal grains and decreased the grain filling rate of the upper grains. At 270 mm irrigation, the PM significantly increased the grain filling rate of the all grains. At 320 mm irrigation, the PM only significantly increased the grain filling rate of the upper grains. The IAA, Z+ZR and ABA content in the grains was positively correlated with the grain weight and grain-filling rates; however, the ETH evolution rate of the grains was negatively correlated with the grain weight and grain-filling rates. These results show that the effect of PM on maize grain filling is related to the irrigation amount and that the grain position on the ear and the grain filling of the upper grains was more sensitive to PM and irrigation than were the other grains. In addition, the PM and irrigation regulated the balance of hormones rather than the content of individual hormones to affect the maize grain filling.