PCK2 maintains intestinal homeostasis and prevents colitis by protecting antibody-secreting cells from oxidative stress.

Maintaining intracellular redox balance is essential for the survival, antibody secretion, and mucosal immune homeostasis of immunoglobulin A (IgA) antibody-secreting cells (ASCs). However, the relationship between mitochondrial metabolic enzymes and the redox balance in ASCs has yet to be comprehensively studied. Our study unveils the pivotal role of mitochondrial enzyme PCK2 in regulating ASCs' redox balance and intestinal homeostasis. We discover that PCK2 loss, whether globally or in B cells, exacerbates dextran sodium sulphate (DSS)-induced colitis due to increased IgA ASC cell death and diminished antibody production. Mechanistically, the absence of PCK2 diverts glutamine into the TCA cycle, leading to heightened TCA flux and excessive mitochondrial reactive oxygen species (mtROS) production. In addition, PCK2 loss reduces glutamine availability for glutathione (GSH) synthesis, resulting in a decrease of total glutathione level. The elevated mtROS and reduced GSH expose ASCs to overwhelming oxidative stress, culminating in cell apoptosis. Crucially, we found that the mitochondria-targeted antioxidant Mitoquinone (Mito-Q) can mitigate the detrimental effects of PCK2 deficiency in IgA ASCs, thereby alleviating colitis in mice. Our findings highlight PCK2 as a key player in IgA ASC survival and provide a potential new target for colitis treatment.

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

Combining Spleen Diameter and the Baveno VI Criteria Assessed by 2-Dimensional Shear Wave Elastography to Rule Out High-Risk Varices.

ABSTRACT: Patients with compensated advanced chronic liver disease (cACLD) can safely spared screening esophagogastroduodenoscopy (EGD) when they meet the Baveno VI criteria as assessed by transient elastography. Recently, the cutoff values of the Baveno VI criteria assessed by 2-dimensional shear wave elastography (2D-SWE) were proposed. We aimed to validate it to rule out high-risk varices (HRVs) in cACLD patients; combine spleen diameter (SPD) with the Baveno VI criteria and assess whether it can spare more screening EGD. A total of 173 cACLD patients with successful liver stiffness (LS) measurements and EGD examinations were included. We analyzed the risk factors that predicted HRVs and compared the performances of different models for ruling out HRVs. The platelet count, LS, and SPD were independent predictors of HRVs. The AUCs of platelet count, LS, spleen stiffness and SPD for diagnosing HRVs were 0.797, 0.757, 0.834, and 0.804, respectively. The Baveno VI criteria assessed by 2D-SWE spared 25.4% of EGD screenings and missed 2.4% of the HRV patients. Combining SPD ≤11.1 cm with the Baveno VI criteria could spare more EGD screenings than just applying the Baveno VI criteria (45.1% vs 25.4%, P < 0.001), and missed 4.9% of the HRV patients. The Baveno VI criteria assessed by 2D-SWE could be safely applied in cACLD patients to rule out HRV patients. The combined model Baveno VI/SPD could safely and significantly increase the rate of spared EGD.

Evaluation of Tumor Fibrosis in Pancreatic Ductal Adenocarcinoma by 2-D Shear Wave Elastography: A Pilot Study.

OBJECTIVE: The aim of the work described here was to investigate the association of the stromal proportion with the elasticity obtained by 2-D shear wave elastography (SWE) and the diagnostic value of elasticity in evaluating tumor stromal fibrosis in pancreatic ductal adenocarcinoma (PDAC). METHODS: Patients who met inclusion criteria underwent pre-operative 2-D SWE examination and intra-operative determination of hardness by palpation from July 2021 to November 2022, and the post-operative specimens were used to evaluate pathological features including the tumor stromal proportion. A receiver operating characteristic curve was created to evaluate its diagnostic value in differentiating the degree of tumor stromal fibrosis. RESULTS: The 2-D SWE measurements in pancreatic lesions were successful in 62 of 69 patients (89.9%). A total of 52 eligible participants were enrolled for subsequent correlation analysis. Elasticity correlated well with tumor stromal proportion (rs = 0.646) and number of tumor cells (rs = -0.585) in PDAC. Moreover, pancreatic elasticity determined by 2-D SWE, palpation-determined hardness and tumor stromal proportion were well correlated with each other. Two-dimensional SWE could clearly distinguish mild and severe stromal fibrosis, and its diagnostic performance was better than that determined by palpation even though the difference was not statistically significant (p = 0.103). CONCLUSION: The elasticity of PDAC obtained using 2-D SWE was closely related to stromal proportion and tumor cellularity and could clearly be used to diagnose the degree of stromal fibrosis, which indicates that 2-D SWE can be a non-invasive predictive imaging biomarker in personalization of therapy and monitoring of treatment.

Tanshinone functions as a coenzyme that confers gain of function of NQO1 to suppress ferroptosis.

Ferroptosis is triggered by the breakdown of cellular iron-dependent redox homeostasis and the abnormal accumulation of lipid ROS. Cells have evolved defense mechanisms to prevent lipid ROS accumulation and ferroptosis. Using a library of more than 4,000 bioactive compounds, we show that tanshinone from Salvia miltiorrhiza (Danshen) has very potent inhibitory activity against ferroptosis. Mechanistically, we found that tanshinone functions as a coenzyme for NAD(P)H:quinone oxidoreductase 1 (NQO1), which detoxifies lipid peroxyl radicals and inhibits ferroptosis both in vitro and in vivo. Although NQO1 is recognized as an oxidative stress response gene, it does not appear to have a direct role in ferroptosis inhibition in the absence of tanshinone. Here, we demonstrate a gain of function of NQO1 induced by tanshinone, which is a novel mechanism for ferroptosis inhibition. Using mouse models of acute liver injury and ischemia/reperfusion heart injury, we observed that tanshinone displays protective effects in both the liver and the heart in a manner dependent on NQO1. Our results link the clinical use of tanshinone to its activity in ferroptosis inhibition.

TET2 deficiency sensitizes tumor cells to statins by reducing HMGCS1 expression.

TET2 (ten-eleven-translocation) protein is a Fe(II)- and α-ketoglutarate-dependent dioxygenase that catalyzes DNA demethylation to regulate gene expression. While TET2 gene is frequently mutated in hematological cancer, its enzymatic activity is also compromised in various solid tumors. Whether TET2 deficiency creates vulnerability for cancer cells has not been studied. Here we reported that TET2 deficiency is associated with the change of lipid metabolism processes in acute myeloid leukemia (AML) patient. We demonstrate that statins, the inhibitors of ß-Hydroxy ß-methylglutaryl-CoA (HMG-CoA) reductase and commonly used cholesterol-lowering medicines, significantly sensitize TET2 deficient tumor cells to apoptosis. TET2 directly regulates the expression of HMG-CoA synthase (HMGCS1) by catalyzing demethylation on its promoter region, and conversely TET2 deficiency leads to significant down-regulation of HMGCS1 expression and the mevalonate pathway. Consistently, overexpression of HMGCS1 in TET2-deficient cells rescues statin-induced apoptosis. We further reveal that decrease of geranylgeranyl diphosphate (GGPP), an intermediate metabolite in the mevalonate pathway, is responsible for statin-induced apoptosis. GGPP shortage abolishes normal membrane localization and function of multiple small GTPases, leading to cell dysfunction. Collectively, our study reveals a vulnerability in TET2 deficient tumor and a potential therapeutic strategy using an already approved safe medicine.

Tumor suppressor CEBPA interacts with and inhibits DNMT3A activity.

DNA methyltransferases (DNMTs) catalyze DNA methylation, and their functions in mammalian embryonic development and diseases including cancer have been extensively studied. However, regulation of DNMTs remains under study. Here, we show that CCAAT/enhancer binding protein α (CEBPA) interacts with the long splice isoform DNMT3A, but not the short isoform DNMT3A2. CEBPA, by interacting with DNMT3A N-terminus, blocks DNMT3A from accessing DNA substrate and thereby inhibits its activity. Recurrent tumor-associated CEBPA mutations, such as preleukemic CEBPAN321D mutation, which is particularly potent in causing AML with high mortality, disrupt DNMT3A association and cause aberrant DNA methylation, notably hypermethylation of PRC2 target genes. Consequently, leukemia cells with the CEBPAN321D mutation are hypersensitive to hypomethylation agents. Our results provide insights into the functional difference between DNMT3A isoforms and the regulation of de novo DNA methylation at specific loci in the genome. Our study also suggests a therapeutic strategy for the treatment of CEBPA-mutated leukemia with DNA-hypomethylating agents.

SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer.

Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid ß-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.