Development and Validation of a Nomogram for Patients Undergoing Transarterial Chemoembolization for Recurrent Hepatocellular Carcinoma After Hepatectomy.

Purpose: This study aims to establish a prognostic nomogram for patients who underwent transarterial chemoembolization (TACE) for recurrent hepatocellular carcinoma (HCC) after hepatectomy. Patients and Methods: Patients who underwent TACE for recurrent early- and middle-stage HCC after hepatectomy between 2009.01 and 2015.12 were included. Enrolled patients were randomly divided into training (n=345) and validation (n=173) cohorts according to a computer-generated randomized number. Independent factors for overall survival (OS) were determined and included in the nomogram based on the univariate and multivariate analyses of the training group. The nomogram was validated and compared to other prognostic models. Discriminative ability and predictive accuracy were determined using the Harrell C index (C-index), area under the receiver operating characteristic curve (AUROC), and calibration curve. Results: The final nomogram was established based on four parameters including resection-to-TACE time interval, recurrent tumor diameter, recurrent tumor number, and AFP level. The C-indexes of the nomogram for predicting OS were 0.67 (95% CI 0.63-0.70) and 0.71 (95% CI 0.68-0.74) in the training and validation cohort respectively. The AUROCs for predicting the 1-year, 2-year and 3-year OS based on the nomogram were also superior to those of the other models. The calibration curve for 3-year survival showed a high congruence between the predicted and actual survival probabilities. According to the scores calculated by the nomogram, patients were stratified into three subgroups: high-risk (scoring ≥53 points), middle-risk (scoring ≥26 and <53 points), and low-risk (scoring <26 points) subgroups with a median OS of 10.1 (95% CI 0.63-0.70), 20.3 (95% CI 17.5-22.5) and 47.0 (95% CI 34.2-59.8) months, respectively. Conclusion: The proposed nomogram served as a new tool to predict individual survival in patients who underwent TACE for recurrent HCC after hepatectomy, with favorable performance and discrimination. For high-risk patients, treatment should be optimized beyond TACE alone based on the nomogram.

Proteomic and metabolomic features in patients with HCC responding to lenvatinib and anti-PD1 therapy.

Combination therapy (lenvatinib/programmed death-1 inhibitor) is effective for treating unresectable hepatocellular carcinoma (uHCC). We reveal that responders have better overall and progression-free survival, as well as high tumor mutation burden and special somatic variants. We analyze the proteome and metabolome of 82 plasma samples from patients with hepatocellular carcinoma (HCC; n = 51) and normal controls (n = 15), revealing that individual differences outweigh treatment differences. Responders exhibit enhanced activity in the alternative/lectin complement pathway and higher levels of lysophosphatidylcholines (LysoPCs), predicting a favorable prognosis. Non-responders are enriched for immunoglobulins, predicting worse outcomes. Compared to normal controls, HCC plasma proteins show acute inflammatory response and platelet activation, while LysoPCs decrease. Combination therapy increases LysoPCs/phosphocholines in responders. Logistic regression/random forest models using metabolomic features achieve good performance in the prediction of responders. Proteomic analysis of cancer tissues unveils molecular features that are associated with side effects in responders receiving combination therapy. In conclusion, our analysis identifies plasma features associated with uHCC responders to combination therapy.

Melitoxin Inhibits Proliferation, Metastasis, and Invasion of Glioma U251 Cells by Down-regulating F2RL1.

As the principal active component of bee venom, melittin has an anti-cancer effect in different cancers. This study was aimed to investigate the effect of melittin in glioma and explore whether F2RL1 is closely involved in glioblastoma cells proliferation. TCGA and GES databases were used to evaluate the role of F2RL1 in gliomas. The U251 cells were divided into a control lentivirus + PBS group (NC-PBS), F2RL1 intervention lentivirus + PBS group (KD-PBS), control lentivirus + melittin group (NC-melittin), and F2RL1 intervention lentivirus + melittin group (KD-melittin). Cell proliferation was detected by MTT and EDU staining assays. The apoptosis rate was assessed by flow cytometry. Expressions of genes related to apoptosis, cycle arrest, migration, and invasion were detected by qRT-PCR. Cellular LDH concentrations were detected by ELISA. The subcutaneous tumor volume of nude mice was analyzed by xenograft method. F2RL1 was significantly overexpressed in glioma tissues and were reduced in the melittin-treated group compared to the blank group. F2RL1 knockdown and melittin alone or in combination increased the proportion of cells in the G1-phase, and the combination was more pronounced. The KD-melittin group showed a decrease in the number of viable cells at 24, 48, 72, and 96 h compared to the NC-PBS group. The number of cell migration and invasion was decreased in the KD-melittin group compared to the other groups. Moreover, the genes related to cell cycle arrest and apoptosis were significantly changed in the KD-melittin group. At weeks 4, 5, and 6, the tumor volume in the KD-melittin group was smaller than that in the KD-PBS group and NC-melittin group. Interference with the target gene F2RL1 inhibited the proliferation of glioma U251 cells, and melittin treatment inhibited the proliferation of glioma U251 cells. Melittin inhibited the proliferation of glioma U251 cells by suppressing the expression of target gene F2RL1.

The Association Between Thymidylate Synthase Gene Polymorphisms and the Risk of Ischemic Stroke in Chinese Han Population.

Family history of hypertension, smoking, diabetes and alcohol consumption and atherosclerotic plaque were identified as common risk factors in IS. We aimed at investigating the relationship between Thymidylate Synthase (TS) gene polymorphisms and ischemic stroke (IS).This case-control research selected and genotyped three single nucleotide polymorphisms (SNPs)of TS( rs699517, rs2790, and rs151264360) with Sanger sequencing in Chinese Han population. We also adopted logistic regression analysis in genetic models for calculating odds ratios and 95% confidence intervals. Genotype-Tissue Expression(GTEx) database analyzed the tissue-specific expression and TS polymorphisms. The ischemic stroke patients showed higher low-density lipoprotein cholesterol and total homocysteine (tHcy). It was found that patients with the TT genotype of rs699517 and GG genotype of rs2790 had larger degrees of tHcy than those with CC + CT genotypes and AA + AG genotypes, respectively. The genotype distribution of the three SNPs did not deviate from Hardy-Weinberg equilibrium (HWE). Haplotype analysis showed that T-G-del was the major haplotype in IS, and C-A-ins was the major haplotype in controls. GTEx database indicated that the rs699517 and rs2790 increased the expression of TS in healthy human and associated with TS expression level in a single tissue. In conclusion: This study has shown that TS rs699517 and rs2790 were significantly related to ischemic stroke patients.

Development of a Combined Oxidative Stress and Endoplasmic Reticulum Stress-Related Prognostic Signature for Hepatocellular Carcinoma.

BACKGROUND: Oxidative stress and endoplasmic reticulum stress are important components of the cellular stress process, which plays a critical role in tumor initiation and progression. METHODS: First, the correlation between oxidative stress and endoplasmic reticulum stress was detected in 68 human hepatocellular carcinoma (HCC) tissue microarray samples by immunohistochemistry. Differentially expressed oxidative stress- and endoplasmic reticulum stressrelated genes (OESGs) then were screened in HCC. Next, an OESGs prognostic signature was constructed for HCC in the training cohort (TCGA-LIHC from The Cancer Genome Atlas), by least absolute shrinkage and selection operator Cox and stepwise Cox regression analyses, and was verified in the external cohort (GSE14520 from the Gene Expression Omnibus). The MCP counter was employed to evaluate immune cell infiltration. The C-index was used to evaluate the predictive power of prognostic signature. Finally, a prognostic nomogram model was constructed to predict the survival probability of patients with HCC based on the results of Cox regression analysis. RESULTS: We demonstrated a positive correlation between oxidative stress and ER stress in human HCC samples. We then identified five OESGs as a prognostic signature consisting of IL18RAP, ECT2, PPARGC1A, STC2, and NQO1 for HCC. Related risk scores correlated with tumor stage, grade, and response to transcatheter arterial chemoembolization therapy, and the higher risk score group had less T cells, CD8+ T cells, cytotoxic lymphocytes and natural killer cell infiltration. The C-index of our OESGs prognostic signature was superior to four previously published signatures. Furthermore, we developed a nomogram based on the OESGs prognostic signature and clinical parameters for patients with HCC that is an effective quantitative analysis tool to predict patient survival. CONCLUSION: The OESGs signature showed excellent performance in predicting survival and therapeutic responses for patients with HCC.

Ten-eleven translocation-2 inactivation restrains IL-10-producing regulatory B cells to enable antitumor immunity in hepatocellular carcinoma.

BACKGROUND AND AIMS: IL-10-producing regulatory B cells (IL-10 + B cells), a dominant regulatory B cell (Breg) subset, foster tumor progression. However, the mechanisms underlying their generation in HCC are poorly understood. Ten-eleven translocation-2 (TET2), a predominant epigenetic regulatory enzyme in B cells, regulates gene expression by catalyzing demethylation of 5-methylcytosine into 5-hydroxymethyl cytosine (5hmC). In this study, we investigated the role of TET2 in IL-10 + B cell generation in HCC and its prospects for clinical application. APPROACH AND RESULTS: TET2 activation in B cells triggered by oxidative stress from the HCC microenvironment promoted IL-10 expression, whereas adoptive transfer of Tet2 -deficient B cells suppressed HCC progression. The aryl hydrocarbon receptor is required for TET2 to hydroxylate Il10 . In addition, high levels of IL-10, TET2, and 5hmc in B cells indicate poor prognosis in patients with HCC. Moreover, we determined TET2 activity using 5hmc in B cells to evaluate the efficacy of anti-programmed death 1 (anti-PD-1) therapy. Notably, TET2 inhibition in B cells facilitates antitumor immunity to improve anti-PD-1 therapy for HCC. CONCLUSIONS: Our findings propose a TET2-dependent epigenetic intervention targeting IL-10 + B cell generation during HCC progression and identify the inhibition of TET2 activity as a promising combination therapy with immune checkpoint inhibitors for HCC.

A Novel Defined RAS-Related Gene Signature for Predicting the Prognosis and Characterization of Biological Function in Osteosarcoma.

Background: Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents with a high incidence and poor prognosis. Activation of the RAS pathway promotes progression and metastasis of osteosarcoma. RAS has been studied in many different tumors; however, the prognostic value of RAS-associated genes in OS remains unclear. On this basis, we investigated the RAS-related gene signature and explored the intrinsic biological features of OS. Methods: We obtained RNA transcriptome sequencing data and clinical information of osteosarcoma patients from the TARGET database. RAS pathway-related genes were obtained from the KEGG pathway database. Molecular subgroups and risk models were developed using consensus clustering and least absolute shrinkage and selection operator (LASSO) regression, respectively. ESTIMATE algorithm and ssGSEA analysis were used to assess the tumor microenvironment and immune penetrance between the two groups. A comprehensive review of gene ontology (GO) and KEGG analyses revealed inherent biological functional differences between the two groups. Results: The consistent clustering showed stratification of osteosarcoma patients into two subtypes based on RAS-associated genes and provided a robust prediction of prognosis. A risk model further confirmed that RAS-related genes are the best prognostic indicators for OS patients. GO analysis showed that GDP/GTP binding, focal adhesion, cytoskeletal motor activity, and cell-matrix junctions were associated with the RAS-related model group. Furthermore, RAS signaling in osteosarcoma based on KEGG analysis was significantly associated with cancer progression, with immune function and tumor microenvironment particularly affected. Conclusion: We constructed a prognostic model founded on RAS-related gene and demonstrated its predictive ability. Then, furtherly exploration of the molecular mechanisms and immune characteristics proved the role of RAS-related gene in the dysregulation in OS.

The Intratumoral Bacterial Metataxonomic Signature of Hepatocellular Carcinoma.

Microbiota is implicated in hepatocellular carcinoma (HCC). The spectrum of intratumoral microbiota associated with HCC progression remains elusive. Fluorescence in situ hybridization revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. Viable anaerobic or aerobic bacteria were recovered in HCC tissues by fresh tissue culture. We performed a comprehensive DNA sequencing of bacterial 16S rRNA genes in 156 samples from 28 normal liver, 64 peritumoral, and 64 HCC tissues, and the DNA sequencing yielded 4.2 million high-quality reads. Both alpha and beta diversity in peritumor and HCC microbiota were increased compared to normal controls. The most predominant phyla in HCC were Patescibacteria, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota. phyla of Proteobacteria, Firmicutes, and Actinobacteriota, and classes of Bacilli and Actinobacteria, were consistently enriched in peritumor and HCC tissues, while Gammaproteobacteria was especially abundant in HCC tissues compared to normal controls. Streptococcaceae and Lactococcus were the marker taxa of HCC cirrhosis. The Staphylococcus branch and Caulobacter branch were selectively enriched in HBV-negative HCCs. The abundance of Proteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteriota, and Saccharimonadia were associated with the clinicopathological features of HCC patients. The inferred functions of different taxa were changed between the microbiota of normal liver and peritumor/HCC. Random forest machine learning achieved great discriminative performance in HCC prediction (area under the curve [AUC] = 1.00 in the training cohort, AUC = 0.950 for top five class signature, and AUC = 0.943 for the top 50 operational taxonomy units [OTUs] in the validation cohort). Our analysis highlights the complexity and diversity of the liver and HCC microbiota and established a specific intratumoral microbial signature for the potential prediction of HCC. IMPORTANCE Gut microbiome is an important regulator of hepatic inflammation, detoxification, and immunity, and contributes to the carcinogenesis of liver cancer. Intratumoral bacteria are supposed to be closer to the tumor cells, forming a microenvironment that may be relevant to the pathological process of hepatocellular carcinoma (HCC). However, the presence of viable intratumoral bacteria remains unclear. It is worth exploring whether the metataxonomic characteristics of intratumoral bacteria can be used as a potential marker for HCC prediction. Here, we present the first evidence of the existence of viable intratumoral bacteria in HCC using the tissue culture method. We revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. We analyzed the diversity, structure, and abundance of normal liver and HCC microbiota. We built a machine learning model for HCC prediction using intratumoral bacterial features. We show that specific taxa represent potential targets for both therapeutic and diagnostic interventions.

Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins.

Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 µg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 µg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.

Extrapolating Prognostic Factors of Primary Curative Resection to Postresection Recurrences Hepatocellular Carcinoma Treatable by Radiofrequency Ablation.

OBJECTIVE: Recurrence after curative resection for hepatocellular carcinoma (HCC) is a major cause of death from this disease. Factors of primary curative resection are available and potential in the prognosis of follow-up treatment. Our aim was to assess the prognostic significance of primary curative resection factors in recurrent HCC patients undergoing radiofrequency ablation therapy (RFA). METHODS: In this retrospective study, we assessed 235 patients who underwent limited RFA of HCC recurrences (tumors ≤ 5 cm; nodules ≤ 3) after primary curative resection. Factors of primary curative resection were collected, and overall survival and recurrence-free survival were evaluated by the Kaplan-Meier method. Univariate and multivariate analyses were used to identify significant prognostic factors. RESULTS: After a median follow-up of 36 months, 54 patients died, and 128 patients had hepatic recurrence. On univariate analyses, patients whose primary tumors were less differentiated (p = 0.032 and p = 0.048) and required less time to recur (p = 0.013 and p = 0.001) after curative resection displayed poorer overall survival and higher recurrence rates following RFA. On multivariate analyses, the pathologic tumor grade (p = 0.026 and p = 0.038) and recurrence-free survival after primary curative resection (p = 0.028 and p < 0.001) emerged as independent risk factors of survival and HCC recurrence. CONCLUSIONS: Primary tumor differentiation and time to recurrence after curative resection are viable prognostic factors of overall survival and further recurrence risk in patients undergoing RFA of recurrent HCC.

Beneficial consequences of Lupeol on middle cerebral artery-induced cerebral ischemia in the rat involves Nrf2 and P38 MAPK modulation.

Lupeol has been reported to exhibit anti-inflammatory and anti-tumor activities in many diseases, but its potential effects in cerebral ischemia injury have not been studied to date. In this work we present evidence for a beneficial effect of lupeol in a rat model of middle cerebral artery occlusion (MCAO) followed by reperfusion (MCAO/R) injury and provide some histological and biochemical evidence for its mechanism of action. A cerebral MCAO rat model was established by vascular occlusion for 2 h, followed by 24 h reperfusion period. The infarct volume, neurological deficits, and brain water content were compared with animals treated during reperfusion with different concentrations of lupeol. Macroscopic parameters, cell viability, pro-inflammatory factors generation, as well as oxidative stress parameters and associated apoptotic signaling cascades were evaluated. Treatment with lupeol significantly reduced the cerebral infarct volume and water content and recovered neuro behavioral functions in affected rats. Lupeol treatment down-regulated the expression of oxidative stress and inflammation factors. In addition, lupeol activated Nrf2, suppressed caspase-3 activity, reduced BAX/Bcl-2 ratio and inhibited phosphorylation of p38 MAPK. The data suggest that lupeol may exert protective effects against cerebral ischemia by suppressing oxidative stress and reduction of inflammation factors possible via activation of nuclear transcription factors and inhibition of cell death pathways.

Adipose-derived mesenchymal stem cells-derived exosome-mediated microRNA-342-5p protects endothelial cells against atherosclerosis.

Exosomes are reported to mediate several disease-related microRNAs (miRNAs) to affect the progression of diseases, including atherosclerosis. Here, we aimed to screen the atherosclerosis-associated miRNAs and preliminarily investigate the potential regulatory mechanism of atherosclerosis. First, the lesion model for human umbilical vein endothelial cells (HUVECs) was favorably constructed. Later, through RNA-sequencing and bioinformatics analyses, miR-342-5p was identified in lesion model for HUVECs. MiR-342-5p overexpression or knockdown evidently promoted or inhibited the apoptosis of HUVECs impaired by H2O2. Mechanistically, PPP1R12B was found to have great potential as a target of miR-342-5p in HUVECs impaired by H2O2, supported by RNA-sequencing and a series of bioinformatics analyses. Meanwhile, the effect of miR-342-5p on PPP1R12B expression in HUVECs' lesion model was explored, revealing that miR-342-5p had an inhibitory role in PPP1R12B expression. Additionally, adipose-derived mesenchymal stem cells (ADSCs) in spindle-like shape and their derived exosomes with 30 to 150 nm diameter were characterized. Furthermore, results showed miR-342-5p was evidently decreased in the presence of ADSCs-derived exosomes. These findings indicated ADSCs-derived exosomes restrained the expression of miR-324-5p in lesion model. Collectively, this work demonstrates an atherosclerosis-associated miR-342-5p and reveals a preliminary possible mechanism in which miR-342-5p mediated by ADSCs-derived exosomes protects endothelial cells against atherosclerosis.