N7-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression.

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.

A TGF-ß-dominant chemoresistant phenotype of hepatoblastoma associated with aflatoxin exposure in children.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common liver cancer in children, posing a serious threat to children's health. Chemoresistance is the leading cause of mortality in patients with HB. A more explicit definition of the features of chemotherapy resistance in HB represents a fundamental urgent need. APPROACH AND RESULTS: We performed an integrative analysis including single-cell RNA sequencing, whole-exome sequencing, and bulk RNA sequencing in 180 HB samples, to reveal genomic features, transcriptomic profiles, and the immune microenvironment of HB. Multicolor immunohistochemistry staining and in vitro experiments were performed for validation. Here, we reported four HB transcriptional subtypes primarily defined by differential expression of transcription factors. Among them, the S2A subtype, characterized by strong expression of progenitor ( MYCN , MIXL1 ) and mesenchymal transcription factors ( TWIST1 , TBX5 ), was defined as a new chemoresistant subtype. The S2A subtype showed increased TGF-ß cancer-associated fibroblast and an immunosuppressive microenvironment induced by the upregulated TGF-ß of HB. Interestingly, the S2A subtype enriched SBS24 signature and significantly higher serum aflatoxin B1-albumin (AFB1-ALB) level in comparison with other subtypes. Functional assays indicated that aflatoxin promotes HB to upregulate TGF-ß. Furthermore, clinical prognostic analysis showed that serum AFB1-ALB is a potential indicator of HB chemoresistance and prognosis. CONCLUSIONS: Our studies offer new insights into the relationship between aflatoxin and HB chemoresistance and provide important implications for its diagnosis and treatment.

Spatial proteomics of immune microenvironment in nonalcoholic steatohepatitis-associated hepatocellular carcinoma.

BACKGROUND AND AIMS: NASH-HCC is inherently resistant to immune checkpoint blockade, but its tumor immune microenvironment is largely unknown. APPROACH AND RESULTS: We applied the imaging mass cytometry to construct a spatially resolved single-cell atlas from the formalin-fixed and paraffin-embedded tissue sections from patients with NASH-HCC, virus-HCC (HBV-HCC and HCV-HCC), and healthy donors. Based on 35 biomarkers, over 750,000 individual cells were categorized into 13 distinct cell types, together with the expression of key immune functional markers. Higher infiltration of T cells, myeloid-derived suppressor cell (MDSCs), and tumor-associated macrophages (TAMs) in HCC compared to controls. The distribution of immune cells in NASH-HCC is spatially heterogeneous, enriched at adjacent normal tissues and declined toward tumors. Cell-cell connections analysis revealed the interplay of MDSCs and TAMs with CD8 + T cells in NASH-HCC. In particular, exhausted programmed cell death 1 (PD-1 + )CD8 + T cells connected with programmed cell death-ligand 1 (PD-L1 + )/inducible T cell costimulator (ICOS + ) MDSCs and TAMs in NASH-HCC, but not in viral HCC. In contrast, CD4 + /CD8 + T cells with granzyme B positivity were reduced in NASH-HCC. Tumor cells expressed low PD-L1 and showed few connections with immune cells. CONCLUSIONS: Our work provides the first detailed spatial map of single-cell phenotypes and multicellular connections in NASH-HCC. We demonstrate that interactions between MDSCs and TAMs with effector T cells underlie immunosuppression in NASH-HCC and are an actionable target.

Long non-coding RNA KB-1460A1.5 promotes ferroptosis by inhibiting mTOR/SREBP-1/SCD1-mediated polyunsaturated fatty acid desaturation in glioma.

Ferroptosis is a new form of regulated cell death caused by the iron-dependent peroxidation of phospholipids and is related to cell metabolism, redox homeostasis and various signalling pathways related to cancer. The long non-coding RNA (lncRNA) KB-1460A1.5 acts as a tumour suppressor gene to regulate tumour growth in gliomas, but its molecular network regulatory mechanism is still unclear. In this study, we found that KB-1460A1.5 can induce ferroptosis in glioma and enhance sensitivity to RSL3, a ferroptosis inducer. Tandem mass tag proteomics and nontargeted metabolomics suggest that KB-1460A1.5 affects polyunsaturated fatty acid metabolic processes. Gas chromatography-mass spectrometry-based medium- and long-chain fatty acid-targeted metabolomics confirmed that upregulation of KB-1460A1.5 decreased the levels of monounsaturated fatty acids, oleic acid (OA) and palmitoleic acid (PO) in glioma cells. The addition of OA and PO restored KB-1460A1.5-induced cellular ferroptosis. Molecularly, KB-1460A1.5 inhibited the mammalian target of rapamycin signalling pathway to suppress the expression of downstream sterol regulatory element-binding protein 1 (SREBP-1), thereby attenuating the stearoyl-CoA desaturase-1 (SCD1)-mediated desaturation of polyunsaturated fatty acids. Finally, an animal model of subcutaneous glioma confirmed that KB-1460A1.5 could inhibit tumour progression, SREBP-1/SCD1 expression and ferroptosis. In conclusion, increasing the expression level of KB-1460A1.5 in glioma can promote the induction of oxidative stress and ferroptosis in cancer cells through SREBP-1/SCD1-mediated adipogenesis, demonstrating therapeutic potential in preclinical models.

FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. METHODS: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. RESULTS: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. CONCLUSIONS: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs. IMPACT AND IMPLICATIONS: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs.

Integrating bioinformatics and experimental validation to unveil disulfidptosis-related lncRNAs as prognostic biomarker and therapeutic target in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) stands as a prevalent malignancy globally, characterized by significant morbidity and mortality. Despite continuous advancements in the treatment of HCC, the prognosis of patients with this cancer remains unsatisfactory. This study aims at constructing a disulfidoptosis­related long noncoding RNA (lncRNA) signature to probe the prognosis and personalized treatment of patients with HCC. METHODS: The data of patients with HCC were extracted from The Cancer Genome Atlas (TCGA) databases. Univariate, multivariate, and least absolute selection operator Cox regression analyses were performed to build a disulfidptosis-related lncRNAs (DRLs) signature. Kaplan-Meier plots were used to evaluate the prognosis of the patients with HCC. Functional enrichment analysis was used to identify key DRLs-associated signaling pathways. Spearman's rank correlation was used to elucidate the association between the DRLs signature and immune microenvironment. The function of TMCC1-AS1 in HCC was validated in two HCC cell lines (HEP3B and HEPG2). RESULTS: We identified 11 prognostic DRLs from the TCGA dataset, three of which were selected to construct the prognostic signature of DRLs. We found that the survival time of low-risk patients was considerably longer than that of high-risk patients. We further observed that the composition and the function of immune cell subpopulations were significantly different between high- and low-risk groups. Additionally, we identified that sorafenib, 5-Fluorouracil, and doxorubicin displayed better responses in the low-score group than those in the high-score group, based on IC50 values. Finally, we confirmed that inhibition of TMCC1-AS1 impeded the proliferation, migration, and invasion of hepatocellular carcinoma cells. CONCLUSIONS: The DRL signatures have been shown to be a reliable prognostic and treatment response indicator in HCC patients. TMCC1-AS1 showed potential as a novel prognostic biomarker and therapeutic target for HCC.

Neutrophil Extracellular Traps Regulate Surgical Brain Injury by Activating the cGAS-STING Pathway.

Surgical brain injury (SBI), induced by neurosurgical procedures or instruments, has not attracted adequate attention. The pathophysiological process of SBI remains sparse compared to that of other central nervous system diseases thus far. Therefore, novel and effective therapies for SBI are urgently needed. In this study, we found that neutrophil extracellular traps (NETs) were present in the circulation and brain tissues of rats after SBI, which promoted neuroinflammation, cerebral edema, neuronal cell death, and aggravated neurological dysfunction. Inhibition of NETs formation by peptidylarginine deiminase (PAD) inhibitor or disruption of NETs with deoxyribonuclease I (DNase I) attenuated SBI-induced damages and improved the recovery of neurological function. We show that SBI triggered the activation of cyclic guanosine monophosphate-adenosine monophosphate synthase stimulator of interferon genes (cGAS-STING), and that inhibition of the cGAS-STING pathway could be beneficial. It is worth noting that DNase I markedly suppressed the activation of cGAS-STING, which was reversed by the cGAS product cyclic guanosine monophosphate-adenosine monophosphate (cGMP-AMP, cGAMP). Furthermore, the neuroprotective effect of DNase I in SBI was also abolished by cGAMP. NETs may participate in the pathophysiological regulation of SBI by acting through the cGAS-STING pathway. We also found that high-dose vitamin C administration could effectively inhibit the formation of NETs post-SBI. Thus, targeting NETs may provide a novel therapeutic strategy for SBI treatment, and high-dose vitamin C intervention may be a promising translational therapy with an excellent safety profile and low cost.

TUBB4B is a novel therapeutic target in non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

Non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) is an emerging malignancy due to the rising prevalence of NAFLD. However, no drug is available to target NAFLD-HCC. In this study, we aim to unravel novel therapeutic targets of NAFLD-HCC utilizing a high-throughput CRISPR/Cas9 screening strategy. We utilized the Epi-drug CRISPR/Cas9 library consisting of single-guide RNAs (sgRNAs) targeting over 1,000 genes representing the FDA-approved drug targets and epigenetic regulators to perform loss-of-function screening in two NAFLD-HCC cell lines (HKCI2 and HKCI10). CRISPR/Cas9 library screening unraveled TUBB4B as an essential gene for NAFLD-HCC cell growth. TUBB4B was overexpressed in NAFLD-HCC tumors compared with adjacent normal tissues (N = 17) and was associated with poor survival (p < 0.01). RNA-sequencing and functional assays revealed that TUBB4B knockout in NAFLD-HCC promoted cell apoptosis, cell cycle arrest, and cellular senescence, leading to suppressed NAFLD-HCC growth in vitro and in vivo. We identified that TUBB4B inhibitor mebendazole (MBZ), an FDA-approved drug, inhibited NAFLD-HCC growth by inducing apoptosis and cellular senescence. Since protein expression of pro-survival Bcl-xL was induced in TUBB4B knockout NAFLD-HCC cells, we examined combination of TUBB4B inhibition with navitoclax, a Bcl-xL inhibitor that selectively targets senescent cells. Consistent with our hypothesis, either TUBB4B knockout or MBZ synergized with navitoclax to inhibit NAFLD-HCC cell growth via the induction of intrinsic and extrinsic apoptosis pathways. In summary, TUBB4B is a novel therapeutic target in NAFLD-HCC. Inhibition of TUBB4B with MBZ in combination with navitoclax synergistically inhibited NAFLD-HCC cell growth, representing a promising strategy for the treatment of NAFLD-HCC. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Targeting N7-methylguanosine tRNA modification blocks hepatocellular carcinoma metastasis after insufficient radiofrequency ablation.

Radiofrequency heat ablation is an ideal radical treatment for hepatocellular carcinoma (HCC). However, insufficient radiofrequency ablation (IRFA) could lead to high recurrence of HCC. N7-methylguanosine (m7G) on tRNAs, an evolutionally conservative modification in mammals and yeast, modulates heat stress responses and tumor progression, while its function in HCC recurrence after IRFA remains unknown. Here, we found that IRFA significantly upregulates the level of m7G tRNA modification and its methyltransferase complex components METTL1/WDR4 in multiple systems including HCC patient-derived xenograft (PDX) mouse, patients' HCC tissues, sublethal-heat-treated models of HCC cell lines, and organoids. Functionally, gain-/loss-of-function assays showed that METTL1-mediated m7G tRNA modification promotes HCC metastasis under sublethal heat exposure both in vitro and in vivo. Mechanistically, we found that METTL1 and m7G tRNA modification enhance the translation of SLUG/SNAIL in a codon frequency-dependent manner under sublethal heat stress. Overexpression of SLUG/SNAIL rescued the malignant potency of METTL1 knockdown HCC cells after sublethal heat exposure. Our study uncovers the key functions of m7G tRNA modification in heat stress responses and HCC recurrence after IRFA, providing molecular basis for targeting METTL1-m7G-SLUG/SNAIL axis to prevent HCC metastasis after radiofrequency heat ablation treatment.

Digital-assisted multidisciplinary treatment for complex occlusal rehabilitation: an 18-month follow-up case report.

BACKGROUND: This case report highlights the importance of standardized and fully digital sequential treatment in complex occlusal rehabilitation cases. To fully resolve the patient's dental needs, such cases often require multidisciplinary interventions including periodontal therapy, endodontic treatment, anterior esthetics, implant restoration, and prosthetic rehabilitation. A fully digital workflow (including facial scanners, intraoral scanners, jaw motion tracking systems, virtual articulators, and computer-aided design software) streamlined the complex treatment, enhancing workflow simplicity, efficiency, visibility, and precision. CASE PRESENTATION: The patient presented with decreased chewing efficiency of the upper and lower prostheses, along with unsatisfactory esthetic appearance of the anterior teeth. After physical examination and radiological assessment, this complex occlusal rehabilitation case required periodontal therapy, anterior esthetic enhancement, implant restoration, and fixed prosthetic rehabilitation. Therefore, a fully digital workflow was adopted. Full-crown prostheses were placed on teeth 13, 23, and 34; a fixed bridge encompassed positions 32 to 42, and single implant crowns were placed on teeth 35 and 36. Implant-supported fixed bridges were constructed for teeth 12 to 22 and 44 to 46, anchored by implants at teeth 12, 22, 44, and 46. All definitive prostheses were fabricated from zirconia ceramics, chosen for their durability and esthetic characteristics. Finally, restorations with satisfactory esthetic and functional characteristics were seated, preserving the tooth and its supporting structures. During treatment and follow-up, the T-scan occlusal analysis system was utilized to continuously monitor and guide the adjustment of occlusal distribution across the patient's dental arches. After 18 months, the patient remains satisfied with the definitive restorations. CONCLUSIONS: This report is intended to help dentists understand and implement standardized and fully digital workflows during the management of complex occlusal rehabilitation cases; it may also facilitate harmonious integration of esthetic and functional characteristics.

Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. DESIGN: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. RESULTS: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. CONCLUSIONS: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts antitumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) reader protein YTHDF1 has been implicated in cancer; however, its role in hepatocellular carcinoma (HCC), especially in non-alcoholic steatohepatitis-associated HCC (NASH-HCC), remains unknown. Here, we investigated the functional role of YTHDF1 in NASH-HCC and its interplay with the tumor immune microenvironment. METHODS: Hepatocyte-specific Ythdf1-overexpressing mice were subjected to a NASH-HCC-inducing diet. Tumor-infiltrating immune cells were profiled with single-cell RNA-sequencing, flow cytometry, and immunostaining. The molecular target of YTHDF1 was elucidated with RNA-sequencing, m6A-sequencing, YTHDF1 RNA immunoprecipitation-sequencing, proteomics, and ribosome-profiling. Ythdf1 in NASH-HCC models was targeted by lipid nanoparticle (LNP)-encapsulated small-interfering Ythdf1. RESULTS: YTHDF1 is overexpressed in tumor tissues compared to adjacent peri-tumor tissues from patients with NASH-HCC. Liver-specific Ythdf1 overexpression drives tumorigenesis in dietary models of spontaneous NASH-HCC. Single-cell RNA-sequencing and flow cytometry revealed that Ythdf1 induced accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed cytotoxic CD8+ T-cell function. Mechanistically, Ythdf1 expression in NASH-HCC cells induced the secretion of IL-6, which mediated MDSC recruitment and activation, leading to CD8+ T-cell dysfunction. EZH2 mRNA was identified as a key YTHDF1 target. YTHDF1 binds to m6A-modified EZH2 mRNA and promotes EZH2 translation. EZH2 in turn increased expression and secretion of IL-6. Ythdf1 knockout synergized with anti-PD-1 treatment to suppress tumor growth in NASH-HCC allografts. Furthermore, therapeutic targeting of Ythdf1 using LNP-encapsulated small-interfering RNA significantly increased the efficacy of anti-PD-1 blockade in NASH-HCC allografts. CONCLUSIONS: We identified that YTHDF1 promotes NASH-HCC tumorigenesis via EZH2-IL-6 signaling, which recruits and activates MDSCs to cause cytotoxic CD8+ T-cell dysfunction. YTHDF1 may be a novel therapeutic target to improve responses to anti-PD-1 immunotherapy in NASH-HCC. IMPACT AND IMPLICATIONS: YTHDF1, a N6-methyladenosine reader, is upregulated in patients with non-alcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC); however, its role in modulating the tumor immune microenvironment in NASH-HCC remains unclear. Here, we show that Ythdf1 mediates immunosuppression in NASH-HCC and that targeting YTHDF1 in combination with immune checkpoint blockade elicits robust antitumor immune responses. Our findings suggest novel therapeutic targets for potentiating the efficacy of immune checkpoint blockade in NASH-HCC and provide the rationale for developing YTHDF1 inhibitors for the treatment of NASH-HCC.

Temozolomide-based sonodynamic therapy induces immunogenic cell death in glioma.

BACKGROUND: In our previous study, we found for the first time that temozolomide (TMZ), the first-line chemotherapeutic agent for glioblastoma (GBM), can generate a large amount of reactive oxygen species (ROS) under ultrasound irradiation. Sonodynamic therapy (SDT) using TMZ as the sonosensitizer produced more potent antitumor effects than TMZ alone. Here, we further evaluate the effects of TMZ-based SDT on subcellular structures and investigate the immunogenic cell death (ICD)-inducing capability of TMZ-based SDT. METHODS: The sonotoxic effects of TMZ were explored in LN229 and GL261 glioma cells. The morphology of endoplasmic reticulum and mitochondria was observed by transmission electron microscopy. The nuclear DNA damage was represented by γ-H2AX staining. Bone marrow-derived dendritic cells (BMDCs) were employed to assess ICD-inducing capability of TMZ-based SDT. A cyclic arginine-glycine-aspartic (c(RGDyC))-modified nanoliposome drug delivery platform was used to improve the tumor targeting of SDT. RESULTS: TMZ-based SDT had a greater inhibitory effect on glioma cells than TMZ alone. Transmission electron microscopy revealed that TMZ-based SDT caused endoplasmic reticulum dilation and mitochondrial swelling. In addition, endoplasmic reticulum stress response (ERSR), nuclear DNA damage and mitochondrial permeability transition pore (mPTP) opening were promoted in TMZ-based SDT group. Most importantly, we found that TMZ-based SDT could promote the "danger signals" produced by glioma cells and induce the maturation and activation of BMDCs, which was associated with the mitochondrial DNA released into the cytoplasm in glioma cells. In vivo experiments showed that TMZ-based SDT could remodel glioma immune microenvironment and provoke durable and powerful anti-tumor immune responses. What's more, the engineered nanoliposome vector of TMZ conferred SDT tumor targeting, providing an option for safer clinical application of TMZ in combination with SDT in the future. CONCLUSIONS: TMZ-based SDT was capable of triggering ICD in glioma. The discovery of TMZ as a sonosensitizer have shown great promise in the treatment of GBM.

The activation of mTOR signalling modulates DNA methylation by enhancing DNMT1 translation in hepatocellular carcinoma.

BACKGROUND: Both dysregulation of mechanistic target of rapamycin (mTOR) signalling and DNA methylation patterns have been shown to be closely associated with tumor progression and serve as promising targets for hepatocellular carcinoma (HCC) therapy. Although their respective roles in HCC have been extensively revealed, the existence of molecular interactions between them remains largely unknown. METHODS: The association of DNA methylation and mTOR signalling in HCC tissues and cell lines was assessed. A Kaplan‒Meier analysis was applied to estimate the overall survival (OS) and recurrence-free survival (RFS) of HCC patients. The modulation of DNMT1 by mTOR in HCC cell lines was determined. The effect of the drug combination in cell lines and mouse models was examined. RESULTS: The results showed that the DNA methylation level was positively associated with the activation of mTOR signalling in HCC tissues and cell lines. Moreover, HCC patients with higher DNA methylation levels and enhanced activation of mTOR signalling exhibited the worst prognosis. Then, we screened methylation-related enzymes and found that the activation of mTOR signalling increased DNMT1 expression and activity. In addition, mTOR enhanced the translational efficiency of DNMT1 in a 4E-BP1-dependent manner, which is based on the pyrimidine rich translational element (PRTE)-containing 5'UTR of DNMT1. Moreover, we demonstrated that the combined inhibition of mTOR and DNMT synergistically inhibited HCC growth in vitro and in vivo. CONCLUSIONS: In addition to some already identified pro-cancer downstream molecules, the activation of mTOR signalling was found to promote DNA methylation by increasing the translation of DNMT1. Furthermore, combined targeting of mTOR and DNMT1 has been demonstrated to have a more effective tumor suppressive function in HCC.

Millihertz magnetic resonance spectroscopy combining the heterodyne readout based on solid-spin sensors.

The sensitivities of quantum sensing in metrology and spectroscopy are drastically influenced by the resolution of the frequency spectrum. However, the resolution is hindered by the decoherence effect between the sensor and the environment. Along these lines, the continue-wave optically detected magnetic resonance (CWODMR) method combined with the heterodyne readout was proposed to break the limitation of the sensor's coherence time. The frequency of the magnetic field was swept to match the unknown signal, and the signal can be transformed to a real-time frequency-domain curve via the heterodyne readout, with a frequency resolution of 4.7 millihertz. Using the nitrogen-vacancy (NV) center ensemble in a diamond as the solid-spin sensors, it was demonstrated that the frequency resolution and precision could be improved proportionally to the low-pass filter parameters of Tc -1 and Tc -1.5, respectively. Furthermore, the introduced method performed the sensing of arbitrary audio signals with a sensitivity of 7.32 nT·Hz-1/2@10 kHz. Our generic approach can be extended to several fields, such as molecular structure determination and biomagnetic field detection, where high-fidelity detection properties across multiple frequency bands are required within small sensing volumes (∼ mm3).

TRPC6 promotes daunorubicin-induced mitochondrial fission and cell death in rat cardiomyocytes with the involvement of ERK1/2-DRP1 activation.

Daunorubicin (DNR-) induced cardiotoxicity seriously restricts its clinical application. Transient receptor potential cation channel subfamily C member 6 (TRPC6) is involved in multiple cardiovascular physiological and pathophysiological processes. However, the role of TRPC6 anthracycline-induced cardiotoxicity (AIC) remains unclear. Mitochondrial fragmentation greatly promotes AIC. TRPC6-mediated ERK1/2 activation has been shown to favor mitochondrial fission in dentate granule cells. The aim of the present study was to elucidate the effects of TRPC6 on daunorubicin- induced cardiotoxicity and identify the mechanisms associated with mitochondrial dynamics. The sparkling results showed that TRPC6 was upregulated in models in vitro and in vivo. TRPC6 knockdown protected cardiomyocytes from DNR-induced cell apoptosis and death. DNR largely facilitated mitochondrial fission, boosted mitochondrial membrane potential collapse and damaged debilitated mitochondrial respiratory function in H9c2 cells，these effects were accompanied by TRPC6 upregulation. siTRPC6 effectively inhibited these mitochondrial adverse aspects showing a positive unexposed effect on mitochondrial morphology and function. Concomitantly, ERK1/2-DRP1 which is related to mitochondrial fission was significantly activated with amplified phosphorylated forms in DNR-treated H9c2 cells. siTRPC6 effectively suppressed ERK1/2-DPR1 over activation, hinting at a potential correlation between TRPC6 and ERK1/2-DRP1 by which mitochondrial dynamics are possibly modulated in AIC. TRPC6 knockdown also raised the Bcl-2/Bax ratio, which may help to block mitochondrial fragmentation-related functional impairment and apoptotic signaling. These findings suggested an essential role of TRPC6 in AIC by intensifying mitochondrial fission and cell death via ERK1/2-DPR1, which could be a potential therapeutic target for AIC.

DNA methylation-regulated LINC02587 inhibits ferroptosis and promotes the progression of glioma cells through the CoQ-FSP1 pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) are considered key players in the formation and development of tumors. Herein, Gene Expression Profiling Interactive Analysis (GEPIA) was employed as a bioinformatics technology. LINC02587 is differentially expressed in bladder urothelial cancer, glioblastoma, lung adenocarcinoma, lung SCC, melanoma, and other tumor tissue and cells. However, its impact on the emergence of glioma and its mechanism is remaining elusive. METHODS: Some of the in vitro assays employed in this study were the CCK-8 / Annexin-V / Transwell assays, colony formation, and wound healing, together with Western blot (WB) evaluation. MSP / BSP assays were employed for assessing the CpG island's methylation status in the LINC02587 promoter. Through transcriptome, ferroptosis-related experiments, and WB evaluation, it was confirmed that LINC02587 is correlated with the regulation of ferroptosis in tumor cells, and CoQ-Fsp1 is one of its regulatory pathways. Moreover, the underlined in-vitro results were further validated by in-vivo studies. RESULTS: The current study shows that the promoter sequence of LINC02587 is regulated by methylation. The silencing of LINC02587 can inhibit cellular proliferative, migrative, and invasive properties, and induce ferroptosis within gliomas through the CoQ-FSP1 pathway. CONCLUSIONS: LINC02587 is likely to be a novel drug target in treating glioma.

Four-dimensional digital design to prediction of the real-time functional rehabilitation in the esthetic zone.

Recent developments in digital technology and materials have improved the accuracy and efficiency of tracking and recording mandibular motion, with various methods being described. The present article describes a digital workflow with complete and accurate 3-dimensional spatial trajectories of mandibular motion to direct the design of lingual restorations. The workflow allowed the lingual curvature of the restoration to conform with the distinctive trajectory of mandibular protrusion.

Insufficient Radiofrequency Ablation Promotes Hepatocellular Carcinoma Metastasis Through N6-Methyladenosine mRNA Methylation-Dependent Mechanism.

BACKGROUND AND AIMS: The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. APPROACH AND RESULTS: Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. CONCLUSIONS: The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.

Non-invasive diagnosis strategy of hepatocellular carcinoma in low-risk population.

AIMS: With prevalence of hepatocellular carcinoma (HCC) in low-risk population (LRP), establishing a non-invasive diagnostic strategy becomes increasingly urgent to spare unnecessary biopsies in this population. The purposes of this study were to find characterisics of HCC and to establish a proper non-invasive method to diagnose HCC in LRP. METHODS: A total of 681 patients in LRP (defined as the population without cirrhosis, chronic HBV infection or HCC history) were collected from 2 institutions. The images of computed tomography (CT) and magnetic resonance imaging (MRI) were manually analysed. We divided the patients into the training cohort (n = 324) and the internal validating cohort (n = 139) by admission time in the first institution. The cohort in the second institution was viewed as the external validation (n = 218). A multivariate logistic regression model incorporating both imaging and clinical independent risk predictors was developed. C-statistics was used to evaluate the diagnostic performance. RESULTS: Besides the major imaging features of HCC (non-rim enhancement, washout and enhancing capsule), tumor necrosis or severe ischemia (TNSI) on imaging and two clinical characteristics (gender and alpha fetoprotein) were also independently associated with HCC diagnosis (all P < 0.01). A clinical model (including 3 major features, TNSI, gender and AFP) was built to diagnose HCC and achieved good diagnostic performance (area under curve values were 0.954 in the training cohort, 0.931 in the internal validation cohort and 0.902 in the external cohort). CONCLUSIONS: The clinical model in this study developed a satisfied non-invasive diagnostic performance for HCC in LRP.