UDP-glucose 6-dehydrogenase (UGDH) produces UDP-α-D-glucuronic acid, the precursors for glycosaminoglycans (GAGs) and proteoglycans of the extracellular matrix. Elevated GAG formation has been implicated in a variety of human diseases, including glioblastoma (GBM). In our previous study, we found that Krüppel-like factor 4 (KLF4) promotes GBM cell migration by binding to methylated DNA, mainly methylated CpGs (mCpG) and transactivating gene expression. We identified UDGH as one of the downstream targets of KLF4-mCpG binding activity. In this study, we show that KLF4 upregulates UGDH expression in a mCpG-dependent manner, and UGDH is required for KLF4-induced cell migration in vitro. UGDH knockdown decreases GAG abundance in GBM cells, as well as cell proliferation and migration in vitro. In intracranial xenografts, reduced UGDH inhibits tumor growth and migration, accompanied by a decrease in the expression of extracellular matrix proteins such as tenascin C, brevican. Our studies demonstrate a novel DNA methylation-dependent UGDH upregulation by KLF4. Developing UGDH antagonists to decrease the synthesis of extracellular matrix components will be a useful strategy for GBM therapy.

Abnormal epigenetic patterns correlate with effector T cell malfunction in tumours1-4, but the cause of this link is unknown. Here we show that tumour cells disrupt methionine metabolism in CD8+ T cells, thereby lowering intracellular levels of methionine and the methyl donor S-adenosylmethionine (SAM) and resulting in loss of dimethylation at lysine 79 of histone H3 (H3K79me2). Loss of H3K79me2 led to low expression of STAT5 and impaired T cell immunity. Mechanistically, tumour cells avidly consumed methionine and outcompeted T cells for methionine by expressing high levels of the methionine transporter SLC43A2. Genetic and biochemical inhibition of tumour SLC43A2 restored H3K79me2 in T cells, thereby boosting spontaneous and checkpoint-induced tumour immunity. Moreover, methionine supplementation improved the expression of H3K79me2 and STAT5 in T cells, and this was accompanied by increased T cell immunity in tumour-bearing mice and patients with colon cancer. Clinically, tumour SLC43A2 correlated negatively with T cell histone methylation and functional gene signatures. Our results identify a mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumour microenvironment. Thus, cancer methionine consumption is an immune evasion mechanism, and targeting cancer methionine signalling may provide an immunotherapeutic approach.

Endothelin-1 (ET-1), an endogenous vasoactive peptide, has been found to play an important role in peripheral pain signaling. Acid-sensing ion channels (ASICs) are key sensors for extracellular protons and contribute to pain caused by tissue acidosis. It remains unclear whether an interaction exists between ET-1 and ASICs in primary sensory neurons. In this study, we reported that ET-1 enhanced the activity of ASICs in rat dorsal root ganglia (DRG) neurons. In whole-cell voltage-clamp recording, ASIC currents were evoked by brief local application of pH 6.0 external solution in the presence of TRPV1 channel blocker AMG9810. Pre-application with ET-1 (1-100 nM) dose-dependently increased the proton-evoked ASIC currents with an EC50 value of 7.42 ± 0.21 nM. Pre-application with ET-1 (30 nM) shifted the concentration-response curve of proton upwards with a maximal current response increase of 61.11% ± 4.33%. We showed that ET-1 enhanced ASIC currents through endothelin-A receptor (ETAR), but not endothelin-B receptor (ETBR) in both DRG neurons and CHO cells co-expressing ASIC3 and ETAR. ET-1 enhancement was inhibited by blockade of G-protein or protein kinase C signaling. In current-clamp recording, pre-application with ET-1 (30 nM) significantly increased acid-evoked firing in rat DRG neurons. Finally, we showed that pharmacological blockade of ASICs by amiloride or APETx2 significantly alleviated ET-1-induced flinching and mechanical hyperalgesia in rats. These results suggest that ET-1 sensitizes ASICs in primary sensory neurons via ETAR and PKC signaling pathway, which may contribute to peripheral ET-1-induced nociceptive behavior in rats.

Glioblastoma (GBM) has limited therapeutic options. DNA repair mechanisms contribute GBM cells to escape therapies and re-establish tumor growth. Multiple studies have shown that POLD2 plays a critical role in DNA replication, DNA repair and genomic stability. We demonstrate for the first time that POLD2 is highly expressed in human glioma specimens and that expression correlates with poor patient survival. siRNA or shRNA POLD2 inhibited GBM cell proliferation, cell cycle progression, invasiveness, sensitized GBM cells to chemo/radiation-induced cell death and reversed the cytoprotective effects of EGFR signaling. Conversely, forced POLD2 expression was found to induce GBM cell proliferation, colony formation, invasiveness and chemo/radiation resistance. POLD2 expression associated with stem-like cell subsets (CD133+ and SSEA-1+ cells) and positively correlated with Sox2 expression in clinical specimens. Its expression was induced by Sox2 and inhibited by the forced differentiation of GBM neurospheres. shRNA-POLD2 modestly inhibited GBM neurosphere-derived orthotopic xenografts growth, when combined with radiation, dramatically inhibited xenograft growth in a cooperative fashion. These novel findings identify POLD2 as a new potential therapeutic target for enhancing GBM response to current standard of care therapeutics.

Endothelin-1 (ET-1), an endogenous vasoconstrictor, has been known as a pro-nociceptive agent involved in multitude of pain. ET-1 acts on endothelin receptors on vascular endothelial cells, sensitizes release of ATP, which then acts on P2X3 receptors on nociceptors and results in mechanical hyperalgesia. Both endothelin receptors and P2X3 receptors are present in primary sensory neuron, where it remains unclear whether there is an interaction between them. Herein, we reported that ET-1 potentiated the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. ET-1 concentration-dependently increased α,β-methylene-ATP (α,β-meATP)-evoked inward currents, which were mediated by P2X3 receptors. ET-1 shifted the α,β-meATP concentration-response curve upwards, with an increase of 34.38 ± 4.72% in the maximal current response to α,β-meATP in the presence of ET-1. ET-1 potentiation of α,β-meATP-evoked currents was voltage-independent. ET-1 potentiated P2X3 receptor-mediated currents through endothelin-A receptors (ETAR), but not endothelin-B receptors (ETBR). ET-1 potentiation was supressed by blockade of intracellular G-protein or protein kinase C (PKC) signaling. Moreover, there is a synergistic effect on mechanical allodynia induced by intraplantar injection of ET-1 and α,β-meATP in rats. Pharmacological blockade of P2X3 receptors also alleviated ET-1-induced mechanical allodynia. These results suggested that ET-1 sensitized P2X3 receptors in primary sensory neurons via an ETAR and PKC signaling pathway. Our data provide evidence that cutaneous ET-1 induced mechanical allodynia not only by increasing the release of ATP from vascular endothelial cells, but also by sensitizing P2X3 receptors on nociceptive DRG neurons.

Major histocompatibility complex-I (MHC-I) presents tumour antigens to CD8+ T cells and triggers anti-tumour immunity. Humans may have 30,000-60,000 long noncoding RNAs (lncRNAs). However, it remains poorly understood whether lncRNAs affect tumour immunity. Here, we identify a lncRNA, lncRNA inducing MHC-I and immunogenicity of tumour (LIMIT), in humans and mice. We found that IFNγ stimulated LIMIT, LIMIT cis-activated the guanylate-binding protein (GBP) gene cluster and GBPs disrupted the association between HSP90 and heat shock factor-1 (HSF1), thereby resulting in HSF1 activation and transcription of MHC-I machinery, but not PD-L1. RNA-guided CRISPR activation of LIMIT boosted GBPs and MHC-I, and potentiated tumour immunogenicity and checkpoint therapy. Silencing LIMIT, GBPs and/or HSF1 diminished MHC-I, impaired antitumour immunity and blunted immunotherapy efficacy. Clinically, LIMIT, GBP- and HSF1-signalling transcripts and proteins correlated with MHC-I, tumour-infiltrating T cells and checkpoint blockade response in patients with cancer. Together, we demonstrate that LIMIT is a cancer immunogenic lncRNA and the LIMIT-GBP-HSF1 axis may be targetable for cancer immunotherapy.

Targeting the p53-MDM2 pathway to reactivate tumor p53 is a chemotherapeutic approach. However, the involvement of this pathway in CD8+ T cell-mediated antitumor immunity is unknown. Here, we report that mice with MDM2 deficiency in T cells exhibit accelerated tumor progression and a decrease in tumor-infiltrating CD8+ T cell survival and function. Mechanistically, MDM2 competes with c-Cbl for STAT5 binding, reduces c-Cbl-mediated STAT5 degradation and enhances STAT5 stability in tumor-infiltrating CD8+ T cells. Targeting the p53-MDM2 interaction with a pharmacological agent, APG-115, augmented MDM2 in T cells, thereby stabilizing STAT5, boosting T cell immunity and synergizing with cancer immunotherapy. Unexpectedly, these effects of APG-115 were dependent on p53 and MDM2 in T cells. Clinically, MDM2 abundance correlated with T cell function and interferon-γ signature in patients with cancer. Thus, the p53-MDM2 pathway controls T cell immunity, and targeting this pathway may treat patients with cancer regardless of tumor p53 status.

Acid-sensing ion channels (ASICs) play an important role in pain associated with tissue acidification. Peripheral inhibitory group II metabotropic glutamate receptors (mGluRs) have analgesic effects in a variety of pain conditions. Whether there is a link between ASICs and mGluRs in pain processes is still unclear. Herein, we show that the group II mGluR agonist LY354740 inhibited acid-evoked ASIC currents and action potentials in rat dorsal root ganglia neurons. LY354740 reduced the maximum current response to protons, but it did not change the sensitivity of ASICs to protons. LY354740 inhibited ASIC currents by activating group II mGluRs. We found that the inhibitory effect of LY354740 was blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analogue 8-Br-cAMP and mimicked by the protein kinase A (PKA) inhibitor H-89. LY354740 also inhibited ASIC3 currents in CHO cells coexpressing mGluR2 and ASIC3 but not in cells expressing ASIC3 alone. In addition, intraplantar injection of LY354740 dose-dependently alleviated acid-induced nociceptive behavior in rats through local group II mGluRs. Together, these results suggested that activation of peripheral group II mGluRs inhibited the functional activity of ASICs through a mechanism that depended on Gi/o proteins and the intracellular cAMP/PKA signaling pathway in rat dorsal root ganglia neurons. We propose that peripheral group II mGluRs are an important therapeutic target for ASIC-mediated pain.

Immune checkpoint blockade (ICB) can produce durable responses against cancer. We and others have found that a subset of patients experiences paradoxical rapid cancer progression during immunotherapy. It is poorly understood how tumors can accelerate their progression during ICB. In some preclinical models, ICB causes hyperprogressive disease (HPD). While immune exclusion drives resistance to ICB, counterintuitively, patients with HPD and complete response (CR) following ICB manifest comparable levels of tumor-infiltrating CD8+ T cells and interferon γ (IFNγ) gene signature. Interestingly, patients with HPD but not CR exhibit elevated tumoral fibroblast growth factor 2 (FGF2) and β-catenin signaling. In animal models, T cell-derived IFNγ promotes tumor FGF2 signaling, thereby suppressing PKM2 activity and decreasing NAD+, resulting in reduction of SIRT1-mediated β-catenin deacetylation and enhanced β-catenin acetylation, consequently reprograming tumor stemness. Targeting the IFNγ-PKM2-β-catenin axis prevents HPD in preclinical models. Thus, the crosstalk of core immunogenic, metabolic, and oncogenic pathways via the IFNγ-PKM2-β-catenin cascade underlies ICB-associated HPD.

Calosoma maximoviczi, a predatory pest beetle, poses a significant threat to wild silk farm production due to its predation on wild silkworms. Given the coexistence of this species with beneficial silkworms in the farm orchards, chemical pesticides are not an ideal solution for controlling its population. In this study, we employed a comprehensive multi-target RNA interference (RNAi) approach to disrupt the olfactory perception of C. maximoviczi through independently silencing 16 odorant receptors (ORs) in the respective genders. Specifically, gene-specific siRNAs were designed to target a panel of ORs, allowing us to investigate the specific interactions between odorant receptors and ligands within this species. Our investigation led to identifying four candidate siOR groups that effectively disrupted the beetle's olfactory tracking of various odorant ligands associated with different trophic levels. Furthermore, we observed sex-specific differences in innate RNAi responses reflected by subsequent gene expression, physiological and behavioral consequences, underscoring the complexity of olfactory signaling and emphasizing the significance of considering species/sex-specific traits when implementing pest control measures. These findings advance our understanding of olfactory coding patterns in C. maximoviczi beetles and establish a foundation for future research in the field of pest management strategies.

The outbreak of coronavirus disease 2019 (COVID-19) posed an unprecedented challenge on public health systems. Despite the measures put in place to contain it, COVID-19 is likely to continue experiencing sporadic outbreaks for some time, and individuals will remain susceptible to recurrent infections. Chimeric antigen receptor (CAR)-T recipients are characterized by durable B-cell aplasia, hypogammaglobulinemia and loss of T-cell diversity, which lead to an increased proportion of severe/critical cases and a high mortality rate after COVID-19 infection. Thus, treatment decisions have become much more complex and require greater caution when considering CAR T-cell immunotherapy. Hence, we reviewed the current understanding of COVID-19 and reported clinical experience in the management of COVID-19 and CAR-T therapy. After a panel discussion, we proposed a rational procedure pertaining to CAR-T recipients with the aim of maximizing the benefit of CAR-T therapy in the post COVID-19 pandemic era.

Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However, it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here, we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models, accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically, PAD4 citrullinates STAT1 at arginine 121, thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1), and the loss of PAD4 abolishes this interaction, ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus, the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target.

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.

Prostaglandin E2 (PGE2) and proton are typical inflammatory mediators. They play a major role in pain processing and hypersensitivity through activating their cognate receptors expressed in terminals of nociceptive sensory neurons. However, it remains unclear whether there is an interaction between PGE2 receptors and proton-activated acid-sensing ion channels (ASICs). Herein, we show that PGE2 enhanced the functional activity of ASICs in rat dorsal root ganglion (DRG) neurons through EP1 and EP4 receptors. In the present study, PGE2 concentration-dependently increased ASIC currents in DRG neurons. It shifted the proton concentration-response curve upwards, without change in the apparent affinity of proton for ASICs. Moreover, PGE2 enhancement of ASIC currents was partially blocked by EP1 or EP4 receptor antagonist. PGE2 failed to enhance ASIC currents when simultaneous blockade of both EP1 and EP4 receptors. PGE2 enhancement was partially suppressed after inhibition of intracellular PKC or PKA signaling, and completely disappeared after concurrent blockade of both PKC and PKA signaling. PGE2 increased significantly the expression levels of p-PKCε and p-PKA in DRG cells. PGE2 also enhanced proton-evoked action potentials in rat DRG neurons. Finally, peripherally administration of PGE2 dose-dependently exacerbated acid-induced nocifensive behaviors in rats through EP1 and EP4 receptors. Our results indicate that PGE2 enhanced the electrophysiological activity of ASICs in DRG neurons and contributed to acidosis-evoked pain, which revealed a novel peripheral mechanism underlying PGE2 involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called "single base editing", and efficiently introduced heterozygous IDH1 R132H mutation (IDH1R132H/WT) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix (ECM), and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1R132H/WT promoted cell migration by upregulating integrin β4 (ITGB4); and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by "single base editing", we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. In summary, the "single base editing" strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.

Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA-mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2(+)CD8(+) T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.

The objectives of this study were to explore physiological and pathological changes in the corneas of diabetic rats by intervening in the expression of silent information regulator 1 (Sirt1) and to investigate whether Sirt1 can regulate the activation of endoplasmic reticulum stress (ERS) while influencing corneal epithelial cell apoptosis under high glucose conditions.

The upregulation of lipid metabolism is considered one of the most common characteristic changes in tumor cells. In this study, the effects of phosphanegold(I) thiolate complexes on the regulation of lipid metabolism in lung cancer cells were investigated. Six novel phosphanegold(I) thiolate complexes capable of inhibiting lung cancer cell growth both in vitro and in vivo were synthesized and characterized. These complexes significantly inhibited the activity of thioredoxin reductases and impaired the normal structure and function of mitochondria, leading to an accumulation of internal reactive oxygen species. In addition, they markedly inhibited key enzymes involved in de novo lipid synthesis, including sterol regulatory element-binding proteins, fatty acid synthase, and adenosine triphosphate citrate lyase, thus reducing endogenous fatty acid and phospholipid synthesis. Both approaches suppressed lipid droplets storage and ultimately induced apoptosis in lung cancer cells. Interestingly, pretreatment with N-acetyl-l-cysteine and free fatty acids partially reversed the inhibitory effect of phosphanegold(I) thiolate complexes on lung cancer cells. These results are the first to indicated that gold(I) complexes are promising candidates for targeting lipid metabolism in lung cancer treatment strategies.

Heat stress during cucumber production often leads to sunburn of leaves, growth retardation of stems and roots, fruit malformation, and even plant death, which have a great impact on the fruit quality and yield. However, no studies on the genetic inheritance and quantitative trait locus mapping of heat tolerance in cucumber at the adult stage have been reported yet. In this study, a set of 86 recombinant inbred lines (RILs) derived from "99281" (heat-tolerant) and "931" (heat-sensitive) were used to identify the heat tolerance QTL in summer 2018, 2019, and 2020. Eight-week-old plants were exposed to a natural high temperature environment in the field, and the heat injury index was used to indicate the heat tolerance performance. Genetic analysis showed that the heat tolerance of adult cucumber is quantitatively inherited. One QTL named qHT1.1 on chromosome 1 was identified. It was delimited by Indel 3-3 and Indel 1-15 and explained 59.6%, 58.1%, and 40.1% of the phenotypic variation in 2018, 2019, and 2020, respectively. The efficiency of marker HT-1, which is closely linked to the locus, was tested using 62 cucumber germplasm accessions and was found to have an accuracy of 97.8% in heat sensitive plants. The qHT1.1 was delimited to a 694.5-kb region, containing 98 genes, nine of which may be involved in heat tolerance. Further sequence analysis showed that there are three single-base substitutions within the coding sequences of Csa1G004990. Gene expression analyses suggested that the expression of Csa1G004990 was significantly higher in "99281" than "931" at 14d, 35d, 42d, and 49d after transplanting. This study provides practically useful markers for heat tolerance breeding in cucumber and provides a basis for further identifying heat tolerant genes.

Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found 2 peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on T cell immunoglobulin and mucin domain containing 4 (Tim-4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FAK family-interacting protein of 200 kDa resulted in Tim-4+ TAM loss via ROS-mediated apoptosis and elevated T cell immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated macrophages positive for complement receptor of the immunoglobulin superfamily (CRIg) were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat patients with ovarian cancer with peritoneal metastasis.