Polystyrene microplastics aggravate radiation-induced intestinal injury in mice.

Radiotherapy is a common treatment for abdominal and pelvic tumors, while the radiation-induced intestinal injury (RIII) is one of the major side-effects of radiotherapy, which reduces the life quality and impedes the treatment completion of cancer patients. Previous studies have demonstrated that environmental pollutant microplastics led to various kinds of injury in the gut, but its effects on RIII are still uncovered. In this study, we fed the C57BL/6J mice with distilled water or 50 µg/d polystyrene microplastics (PSMPs) for 17 days and exposed the mice to total abdominal irradiation (TAI) at day 14. Then the severity of RIII was examined by performing histopathological analysis and microbial community analysis. The results demonstrated that PSMPs significantly aggravated RIII in small intestine rather than colon of mice upon TAI. PSMPs increased levels of the histopathological damage and the microbial community disturbance in mice small intestine, shown by the overabundance of Akkermansiaceae and the decrease of microflora including Lactobacillaceae, Muribaculaceae and Bifidobacteriaceae. In conclusion, our results suggested that more microplastics exposure might led to more severe RIII, which should be considered in patients' daily diet adjustment and clinical radiotherapy plan evaluation. Furthermore, this study also called for the further researches to uncover the underlying mechanism and develop novel strategies to attenuate RIII in mice intestine.

Study on the Nodal Composite Bearing Performance of Nontruncated PHC Pipe Pile and Bearing Platform.

In this paper, low circumferential reciprocating load foot-scale tests were performed on two nontruncated PHC B 600 130 tubular piles with bearing nodes to characterize the damage process and morphology of the specimens and to investigate the load-carrying performance of the members. The test results reveal that under the action of tensile-bending-shear loading, the bearing concrete in the node area buckles and is damaged, the anchored reinforcement in the node area yields, the constraint is weakened, an articulation point is formed, and the node rotational capacity increases. When the embedment depth increases from 200 mm to 300 mm, the ultimate bearing capacities of the positive and negative nodes increase by 31.04% and 36.16%, respectively. A numerical simulation is used to verify the test results. Considering the four types of piles without truncated nodes, the numerical simulation is used to analyze the node-bearing capacity at different embedment depths. Finally, a preferred node type is proposed as follows: a terminal plate welded anchor bar and pipe pile core-filled longitudinal reinforcement anchored into the bearing node, with a preferred embedment depth of 250 mm.

Single-cell transcriptomics reveal the microenvironment landscape of perfluorooctane sulfonate-induced liver injury in female mice.

Epidemic and animal studies have reported that perfluoroalkyl and polyfluoroalkyl substances (PFASs) are strongly associated with liver injury; however, to date, the effects of PFASs on the hepatic microenvironment remain largely unknown. In this study, we established perfluorooctane sulfonic acid (PFOS)-induced liver injury models by providing male and female C57BL/6 mice with water containing PFOS at varying doses for 4 weeks. Hematoxylin and eosin staining revealed that PFOS induced liver injury in both sexes. Elevated levels of serum aminotransferases including those of alanine aminotransferase and aspartate transaminase were detected in the serum of mice treated with PFOS. Female mice exhibited more severe liver injury than male mice. We collected the livers from female mice and performed single-cell RNA sequencing. In total, 36,529 cells were included and grouped into 10 major cell types: B cells, granulocytes, T cells, NK cells, monocytes, dendritic cells, macrophages, endothelial cells, fibroblasts, and hepatocytes. Osteoclast differentiation was upregulated and the T cell receptor signaling pathway was significantly downregulated in PFOS-treated livers. Further analyses revealed that among immune cell clusters in PFOS-treated livers, Tcf7+CD4+T cells were predominantly downregulated, whereas conventional dendritic cells and macrophages were upregulated. Among the fibroblast subpopulations, hepatic stellate cells were significantly enriched in PFOS-treated female mice. CellphoneDB analysis suggested that fibroblasts interact closely with endothelial cells. The major ligand-receptor pairs between fibroblasts and endothelial cells in PFOS-treated livers were Dpp4_Cxcl12, Ackr3_Cxcl12, and Flt1_complex_Vegfa. These genes are associated with directing cell migration and angiogenesis. Our study provides a general framework for understanding the microenvironment in the livers of female mice exposed to PFOS at the single-cell level.

Single-cell Transcriptomic Analysis Reveals an Immunosuppressive Network Between POSTN CAFs and ACKR1 ECs in TKI-resistant Lung Cancer.

BACKGROUND/AIM: Tyrosine kinase inhibitor (TKI) therapy, a principal treatment for advanced non-small cell lung cancer (NSCLC), frequently encounters the development of drug resistance. The tumor microenvironment (TME) plays a critical role in the progression of NSCLC, yet the relationship between endothelial cells (ECs) and cancer-associated fibroblasts (CAFs) subpopulations in TKI treatment resistance remains largely unexplored. MATERIALS AND METHODS: The BioProject database PRJNA591860 project was used to analyze scRNA-seq data including 49 advanced-stage NSCLC samples across three different time points: pre-targeted therapy (naïve), post-partial response (PR) to targeted therapy, and post-progressive disease (PD) stage. The data involved clustering stromal cells into multiple CAFs and ECs subpopulations. The abundance changes and functions of each cluster during TKI treatment were investigated by KEGG and GO analysis. Additionally, we identified specific transcription factors and metabolic pathways via DoRothEA and scMetabolism. Moreover, cell-cell communications between PD and PR stages were compared by CellChat. RESULTS: ECs and CAFs were clustered and annotated using 49 scRNA-seq samples. We identified seven ECs subpopulations, with OIT3 ECs showing enrichment in the PR phase with a drug-resistance phenotype, and ACKR1 ECs being prevalent in the PD phase with enhanced cell adhesion. Similarly, CAFs were clustered into 7 subpopulations. PLA2G2A CAFs were predominant in PR, whereas POSTN CAFs were prevalent in PD, characterized by an immunomodulatory phenotype and increased collagen secretion. CellChat analysis showed that ACKR1 ECs strongly interacted with macrophage through the CD39 pathway and POSTN CAFs secreted Tenascin-C (TNC) to promote the progression of epithelial cells, primarily malignant ones, in PD. CONCLUSION: This study reveals that POSTN CAFs and ACKR1 ECs are associated with resistance to TKI treatment, based on single-cell sequencing.

Botrytis cinerea hypovirulent strain △BcSpd1 induced Panax ginseng defense.

Background: Gray mold, caused by Botrytis cinerea, is one of the major fungal diseases in agriculture. Biological methods are preferred over chemical fungicides to control gray mold since they are less toxic to the environment and could induce the resistance to pathogens in plants. In this work, we try to understand if ginseng defense to B. cinerea could be induced by fungal hypovirulent strain △BcSpd1. BcSpd1 encodes Zn(II)2Cys6 transcription factor which regulates fungal pathogenicity and we recently reported △BcSpd1 mutants reduced fungal virulence. Methods: We performed transcriptomic analysis of the host to investigate the induced defense response of ginseng treated by B. cinerea △BcSpd1. The metabolites in ginseng flavonoids pathway were determined by UPLC-ESI-MS/MS and the antifungal activates were then performed. Results: We found that △BcSpd1 enhanced the ginseng defense response when applied to healthy ginseng leaves and further changed the metabolism of flavonoids. Compared with untreated plants, the application of △BcSpd1 on ginseng leaves significantly increased the accumulation of p-coumaric acid and myricetin, which could inhibit the fungal growth. Conclusion: B. cinerea △BcSpd1 could effectively induce the medicinal plant defense and is referred to as the biological control agent in ginseng disease management.

Causal Factor Disentanglement for Few-Shot Domain Adaptation in Video Prediction.

An important challenge in machine learning is performing with accuracy when few training samples are available from the target distribution. If a large number of training samples from a related distribution are available, transfer learning can be used to improve the performance. This paper investigates how to do transfer learning more effectively if the source and target distributions are related through a Sparse Mechanism Shift for the application of next-frame prediction. We create Sparse Mechanism Shift-TempoRal Intervened Sequences (SMS-TRIS), a benchmark to evaluate transfer learning for next-frame prediction derived from the TRIS datasets. We then propose to exploit the Sparse Mechanism Shift property of the distribution shift by disentangling the model parameters with regard to the true causal mechanisms underlying the data. We use the Causal Identifiability from TempoRal Intervened Sequences (CITRIS) model to achieve this disentanglement via causal representation learning. We show that encouraging disentanglement with the CITRIS extensions can improve performance, but their effectiveness varies depending on the dataset and backbone used. We find that it is effective only when encouraging disentanglement actually succeeds in increasing disentanglement. We also show that an alternative method designed for domain adaptation does not help, indicating the challenging nature of the SMS-TRIS benchmark.

Transcriptome and metabolite analyses indicated the underlying molecular responses of Asian ginseng (Panax ginseng) toward Colletotrichum panacicola infection.

Panax ginseng Meyer is one of the most valuable plants and is widely used in China, while ginseng anthracnose is one of the most destructive diseases. Colletotrichum panacicola could infect ginseng leaves and stems and causes serious anthracnose disease, but its mechanism is still unknown. Here, transcriptome and metabolism analyses of the host leaves were conducted to investigate the ginseng defense response affected by C. panacicola. Upon C. panacicola infection, ginseng transcripts altered from 14 to 24 h, and the expression of many defense-related genes switched from induction to repression. Consequently, ginseng metabolites in the flavonoid pathway were changed. Particularly, C. panacicola repressed plant biosynthesis of the epicatechin and naringin while inducing plant biosynthesis of glycitin, vitexin/isovitexin, and luteolin-7-O-glucoside. This work indicates C. panacicola successful infection of P. ginseng by intervening in the transcripts of defense-related genes and manipulating the biosynthesis of secondary metabolites, which might have antifungal activities.

Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation.

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H- but not K8-K393R-induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C-mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

Targeting NAD+ metabolism of hepatocellular carcinoma cells by lenvatinib promotes M2 macrophages reverse polarization, suppressing the HCC progression.

BACKGROUND: Lowered nicotinamide adenine dinucleotide (NAD+) levels in tumor cells drive tumor hyperprogression during immunotherapy, and its restoration activates immune cells. However, the effect of lenvatinib, a first-line treatment for unresectable hepatocellular carcinoma (HCC), on NAD+ metabolism in HCC cells, and the metabolite crosstalk between HCC and immune cells after targeting NAD+ metabolism of HCC cells remain unelucidated. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography multiple reaction monitoring-mass spectrometry (UHPLC-MRM-MS) were used to detect and validate differential metabolites. RNA sequencing was used to explore mRNA expression in macrophages and HCC cells. HCC mouse models were used to validate the effects of lenvatinib on immune cells and NAD+ metabolism. The macrophage properties were elucidated using cell proliferation, apoptosis, and co-culture assays. In silico structural analysis and interaction assays were used to determine whether lenvatinib targets tet methylcytosine dioxygenase 2 (TET2). Flow cytometry was performed to assess changes in immune cells. RESULTS: Lenvatinib targeted TET2 to synthesize and increase NAD+ levels, thereby inhibiting decomposition in HCC cells. NAD+ salvage increased lenvatinib-induced apoptosis of HCC cells. Lenvatinib also induced CD8+ T cells and M1 macrophages infiltration in vivo. And lenvatinib suppressed niacinamide, 5-Hydroxy-L-tryptophan and quinoline secretion of HCC cells, and increased hypoxanthine secretion, which contributed to proliferation, migration and polarization function of macrophages. Consequently, lenvatinib targeted NAD+ metabolism and elevated HCC-derived hypoxanthine to enhance the macrophages polarization from M2 to M1. Glycosaminoglycan binding disorder and positive regulation of cytosolic calcium ion concentration were characteristic features of the reverse polarization. CONCLUSIONS: Targeting HCC cells NAD+ metabolism by lenvatinib-TET2 pathway drives metabolite crosstalk, leading to M2 macrophages reverse polarization, thereby suppressing HCC progression. Collectively, these novel insights highlight the role of lenvatinib or its combination therapies as promising therapeutic alternatives for HCC patients with low NAD+ levels or high TET2 levels.

Case report: Abolishing primary resistance to PD-1 blockade by short-term treatment of lenvatinib in a patient with advanced metastatic renal cell carcinoma.

Anti-PD-1 immunotherapy has been extensively used in treatment of patients with advanced metastatic renal cell carcinoma (mRCC). Several prospective clinical trials showed that the combined treatment of anti-PD-1 antibody plus lenvatinib, a potent receptor tyrosine kinase inhibitor (TKI), exhibited high response rate compared with single-agent sunitinib. However, whether the patients with primary resistance to PD-1 blockade could benefit from the addition of lenvatinib is still unclear. Herein, we reported a patient with mRCC who was primary resistant to pembrolizumab and achieved a durable complete response after a short-term treatment with lenvatinib. This case report indicates that the patients with primary resistance to anti-PD-1 therapy could benefit from the short-term lenvatinib in combination with anti-PD-1 therapy, and provides a useful paradigm worthy of establishing a clinical trial for mRCC patients with primary resistance to anti-PD-1 therapy.

An fMRI Dataset for Concept Representation with Semantic Feature Annotations.

The neural representation of concepts is a focus of many cognitive neuroscience studies. Prior works studying concept representation with neural imaging data have been largely limited to concrete concepts. The use of relatively small and constrained sets of stimuli leaves open the question of whether the findings can generalize other concepts. We share an fMRI dataset in which 11 participants thought of 672 individual concepts, including both concrete and abstract concepts. The concepts were probed using words paired with images in which the words were selected to cover a wide range of semantic categories. Furthermore, according to the componential theories of concept representation, we collected the 54 semantic features of the 672 concepts comprising sensory, motor, spatial, temporal, affective, social, and cognitive experiences by crowdsourcing annotations. The quality assessment results verify this as a high-quality neuroimaging dataset. Such a dataset is well suited to study how the brain represents different semantic features and concepts, creating the essential condition to investigate the neural representation of individual concepts.

UBE2T-mediated Akt ubiquitination and Akt/ß-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism.

The oncogene protein ubiquitin-conjugating enzyme E2T (UBE2T) is reported to be upregulated in hepatocellular carcinoma (HCC) and correlated with poor clinical outcomes of HCC patients. However, the underlying mechanism by which UBE2T exerts its oncogenic function in HCC remains largely unexplored. In this study, in vitro and in vivo experiments suggested that UBE2T promoted HCC development including proliferation and metastasis. GSEA analysis indicated that UBE2T was positively correlated with pyrimidine metabolism, and LC/MS-MS metabolomics profiling revealed that the key products of pyrimidine metabolism were significantly increased in UBE2T-overexpressing cells. UBE2T overexpression led to the upregulation of several key enzymes catalyzing de novo pyrimidine synthesis, including CAD, DHODH, and UMPS. Moreover, the utilization of leflunomide, a clinically approved DHODH inhibitor, blocked the effect of UBE2T in promoting HCC progression. Mechanistically, UBE2T increased Akt K63-mediated ubiquitination and Akt/ß-catenin signaling pathway activation. The disruption of UBE2T-mediated ubiquitination on Akt, including E2-enzyme-deficient mutation (C86A) of UBE2T and ubiquitination-site-deficient mutation (K8/14 R) of Akt impaired UBE2T's effect in upregulating CAD, DHODH, and UMPS. Importantly, we demonstrated that UBE2T was positively correlated with p-Akt, ß-catenin, CAD, DHODH, and UMPS in HCC tumor tissues. In summary, our study indicates that UBE2T increases pyrimidine metabolism by promoting Akt K63-linked ubiquitination, thus contributing to HCC development. This work provides a novel insight into HCC development and a potential therapeutic strategy for HCC patients.

Modeling and Control of COVID-19 Transmission from a Perspective of Polymerization Reaction Dynamics.

Due to the serious economic losses and deaths caused by COVID-19, the modeling and control of such a pandemic has become a hot research topic. This paper finds an analogy between a polymerization reaction and COVID-19 transmission dynamics, which will provide a novel perspective to optimal control measures. Susceptible individuals, exposed people, infected cases, recovered population, and the dead can be assumed to be specific molecules in the polymerization system. In this paper, a hypothetical polymerization reactor is constructed to describe the transmission of an epidemic, and its kinetic parameters are regressed by the least-squares method. The intensity of social distancing u is considered to the mixing degree of the reaction system, and contact tracing and isolation ρ can be regarded as an external circulation in the main reactor to reduce the concentration of active species. Through these analogies, this model can predict the peak infection, deaths, and end time of the epidemic under different control measures to support the decision-making process. Without any measures (u = 1.0 and ρ = 0), more than 90% of the population would be infected. It takes several years to complete herd immunity by nonpharmacological intervention when the proportion of deaths is limited to less than 5%. However, vaccination can reduce the time to tens to hundreds of days, which is related to the maximum number of vaccines per day.

Wnt/ß-catenin inhibitor ICG-001 enhances the antitumor efficacy of radiotherapy by increasing radiation-induced DNA damage and improving tumor immune microenvironment in hepatocellular carcinoma.

BACKGROUND AND PURPOSE: Radiotherapy (RT) has a promising anti-tumor effect depending on its effects on both cancer cells and tumor immune microenvironment (TIME). As one of the most common alterations in hepatocellular carcinoma (HCC), wnt/ß-catenin pathway activation, has been reported to induce radioresistance and suppressive TIME. In this study, we aim to explore the effect of wnt/ß-catenin inhibitor ICG-001 on radiosensitivity and RT-related TIME of HCC and the underlying mechanism. MATERIALS AND METHODS: C57BL/6 and nude mouse tumor models were used to evaluate the efficacy of different treatments on tumor growth, recurrence and mice survival. Flow cytometry was performed to assess tumor infiltrating lymphocytes (TILs). DNA damage response (DDR) and radioresistance was investigated by colony formation assays, γ-H2AX and micronuclei measurements. RESULTS: The addition of ICG-001 to RT exhibited better anti-tumor and survival-prolong efficacy in C57BL/6 than nude mice. TILs analysis revealed that ICG-001 plus RT boosted the infiltration and IFN-γ production of TIL CD8+ T cells, meanwhile reduced the number of Tregs. Moreover, mechanistic study demonstrated that ICG-001 increased the radiation-induced DDR of HCC cells by suppressing p53, thus leading to stronger activation of cGAS/STING pathway. Utilization of cGAS/STING pathway inhibitors impaired the therapeutic effect of combination therapy. Furthermore, combination therapy led to stronger immunologic memory and tumor relapse prevention. CONCLUSIONS: Our findings showed that ICG-001 displayed both local and systematic effects by increasing radiosensitivity and improving immunity in HCC, which indicated that ICG-001 might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in HCC patients.

A new model for COVID-19 control and its implementation in the city of Honghu, China: a case report.

BACKGROUND: Novel coronavirus pneumonia has been the most serious worldwide public health emergency since being identified in December 2019. The rapid spread of the pandemic and the strong human to human infection rate of COVID-19 poses a great prevention challenge. There has been an explosion in the number of confirmed cases in several cities near Wuhan, including the highest in Honghu, Jinzhou. Owing to the limited admission capacity and medical resources, increasing numbers of suspected cases of COVID-19 infection were difficult to confirm or treat. CASE PRESENTATION: Following the arrival of the Guangdong medical aid team on 11 February, 2020, COVID-19 care in Honghu saw changes after a series of solutions were implemented based on the 'Four-Early' and 'Four-centralization' management measures. The 'Four-Early' measures are: early detection, early reporting, early quarantine, and early treatment for meeting an urgent need like the COVID-19 pandemic. 'Four-centralization' refers to the way in which recruited medical teams can make full use of medical resources to give patients the best treatment. These solutions successfully increased the recovery rate and reduced mortality among patients with COVID-19 in Honghu. CONCLUSIONS: This management strategy is called the 'Honghu Model' which can be generalized to enable the prevention and management of COVID-19 worldwide.

Deep learning enables discovery of highly potent anti-osteoporosis natural products.

A pre-trained self-attentive message passing neural network (P-SAMPNN) model was developed based on our anti-osteoclastogenesis dataset for virtual screening purpose. Validation processes proved that P-SAMPNN model was significantly superior to the other base line models. A commercially available natural product library was virtually screened by the P-SAMPNN model and resulted in confirmed 5 hits from 10 selected virtual hits. Among the confirmed hits, compounds AP-123/40765213 and AE-562/43462182 are the nanomolar inhibitors against osteoclastogenesis with a new scaffold. Further studies indicate that AP-123/40765213 and AE-562/43462182 significantly suppress the mRNA expression of RANK and downregulate the expressions of osteoclasts-related genes Ctsk, Nfatc1, and Tracp. Our work demonstrated that P-SAMPNN method can guide phenotype-based drug discovery.

Neural Encoding and Decoding With Distributed Sentence Representations.

Building computational models to account for the cortical representation of language plays an important role in understanding the human linguistic system. Recent progress in distributed semantic models (DSMs), especially transformer-based methods, has driven advances in many language understanding tasks, making DSM a promising methodology to probe brain language processing. DSMs have been shown to reliably explain cortical responses to word stimuli. However, characterizing the brain activities for sentence processing is much less exhaustively explored with DSMs, especially the deep neural network-based methods. What is the relationship between cortical sentence representations against DSMs? What linguistic features that a DSM catches better explain its correlation with the brain activities aroused by sentence stimuli? Could distributed sentence representations help to reveal the semantic selectivity of different brain areas? We address these questions through the lens of neural encoding and decoding, fueled by the latest developments in natural language representation learning. We begin by evaluating the ability of a wide range of 12 DSMs to predict and decipher the functional magnetic resonance imaging (fMRI) images from humans reading sentences. Most models deliver high accuracy in the left middle temporal gyrus (LMTG) and left occipital complex (LOC). Notably, encoders trained with transformer-based DSMs consistently outperform other unsupervised structured models and all the unstructured baselines. With probing and ablation tasks, we further find that differences in the performance of the DSMs in modeling brain activities can be at least partially explained by the granularity of their semantic representations. We also illustrate the DSM's selectivity for concept categories and show that the topics are represented by spatially overlapping and distributed cortical patterns. Our results corroborate and extend previous findings in understanding the relation between DSMs and neural activation patterns and contribute to building solid brain-machine interfaces with deep neural network representations.

UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation.

BACKGROUND: Radioresistance is the major obstacle in radiation therapy (RT) for hepatocellular carcinoma (HCC). Dysregulation of DNA damage response (DDR), which includes DNA repair and cell cycle checkpoints activation, leads to radioresistance and limits radiotherapy efficacy in HCC patients. However, the underlying mechanism have not been clearly understood. METHODS: We obtained 7 pairs of HCC tissues and corresponding non-tumor tissues, and UBE2T was identified as one of the most upregulated genes. The radioresistant role of UBE2T was examined by colony formation assays in vitro and xenograft tumor models in vivo. Comet assay, cell cycle flow cytometry and γH2AX foci measurement were used to investigate the mechanism by which UBE2T mediating DDR. Chromatin fractionation and immunofluorescence staining were used to assess cell cycle checkpoint kinase 1(CHK1) activation. Finally, we analyzed clinical data from HCC patients to verify the function of UBE2T. RESULTS: Here, we found that ubiquitin-conjugating enzyme E2T (UBE2T) was upregulated in HCC tissues, and the HCC patients with higher UBE2T levels exhibited poorer outcomes. Functional studies indicated that UBE2T increased HCC radioresistance in vitro and in vivo. Mechanistically, UBE2T-RNF8, was identified as the E2-E3 pair, physically bonded with and monoubiquitinated histone variant H2AX/γH2AX upon radiation exposure. UBE2T-regulated H2AX/γH2AX monoubiquitination facilitated phosphorylation of CHK1 for activation and CHK1 release from the chromatin to cytosol for degradation. The interruption of UBE2T-mediated monoubiquitination on H2AX/γH2AX, including E2-enzyme-deficient mutation (C86A) of UBE2T and monoubiquitination-site-deficient mutation (K119/120R) of H2AX, cannot effectively activate CHK1. Moreover, genetical and pharmacological inhibition of CHK1 impaired the radioresistant role of UBE2T in HCC. Furthermore, clinical data suggested that the HCC patients with higher UBE2T levels exhibited worse response to radiotherapy. CONCLUSION: Our results revealed a novel role of UBE2T-mediated H2AX/γH2AX monoubiquitination on facilitating cell cycle arrest activation to provide sufficient time for radiation-induced DNA repair, thus conferring HCC radioresistance. This study indicated that disrupting UBE2T-H2AX-CHK1 pathway maybe a promising potential strategy to overcome HCC radioresistance.

ATR inhibitor AZD6738 enhances the antitumor activity of radiotherapy and immune checkpoint inhibitors by potentiating the tumor immune microenvironment in hepatocellular carcinoma.

BACKGROUND: Radioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation leads to the inhibition of immune microenvironment, thus impairing the antitumor effect of radioimmunotherapy. However, it is unclear whether inhibition of the DDR pathway can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of DDR inhibitor AZD6738 on the combination of radiotherapy and immune checkpoint inhibitors (ICIs) in HCC. METHODS: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control and tumor recurrence inhibition. Effects of each treatment regimen on the alterations of immunophenotypes including the quantification, activation, proliferating ability, exhaustion marker expression, and memory status were assessed by flow cytometry. RESULTS: AZD6738 further increased radiotherapy-stimulated CD8+ T cell infiltration and activation and reverted the immunosuppressive effect of radiation on the number of Tregs in mice xenografts. Moreover, compared with radioimmunotherapy (radiotherapy plus anti-PD-L1 (Programmed death ligand 1)), the addition of AZD6738 boosted the infiltration, increased cell proliferation, enhanced interferon (IFN)-γ production ability of TIL (tumor-infiltrating lymphocyte) CD8+ T cells, and caused a decreasing trend in the number of TIL Tregs and exhausted T cells in mice xenografts. Thus, the tumor immune microenvironment was significantly improved. Meanwhile, triple therapy (AZD6738 plus radiotherapy plus anti-PD-L1) also induced a better immunophenotype than radioimmunotherapy in mice spleens. As a consequence, triple therapy displayed greater benefit in antitumor efficacy and mice survival than radioimmunotherapy. Mechanism study revealed that the synergistic antitumor effect of AZD6738 with radioimmunotherapy relied on the activation of cyclic GMP-AMP synthase /stimulator of interferon genes (cGAS/STING) signaling pathway. Furthermore, triple therapy led to stronger immunologic memory and lasting antitumor immunity than radioimmunotherapy, thus preventing tumor recurrence in mouse models. CONCLUSIONS: Our findings indicate that AZD6738 might be a potential synergistic treatment for radioimmunotherapy to control the proliferation of HCC cells, prolong survival, and prevent tumor recurrence in patients with HCC by improving the immune microenvironment.