Integration of Single-Cell RNA Sequencing and Bulk RNA Sequencing to Identify an Immunogenic Cell Death-Related 5-Gene Prognostic Signature in Hepatocellular Carcinoma.

Introduction: Immunogenic cell death (ICD) can enhance the potency of immunotherapy in cancer treatment. Nevertheless, it is ambiguous how ICD-related genes (ICDRGs) contribute to hepatocellular carcinoma (HCC). Methods: Single-cell RNA sequencing (scRNA-seq) data were used to distinguish malignant cells from normal cells in the HCC tumor microenvironment(TME). Bulk RNA sequencing data was employed to acquire the landscape of the 33 ICDRGs. Unsupervised clustering identified two ICD molecular subtypes. The cellular infiltration characteristics and biological behavior in different subtypes were analyzed by ssGSEA. Subsequently, differentially expressed genes (DEGs) between the two subtypes were determined, based on which patients were classified into three gene clusters. Then, the prognostic model was constructed by Lasso-Cox analysis. Finally, we investigated the expression of risk genes in cancer cell line encyclopedia (CCLE) and validated the function of NKX3-2 in vitro experiments. Results: ICD scores and ICDRGs expression in malignant cells were significantly lower than in normal cells by scRNA-seq analysis. ICD-high subtype was characterized by ICD-related gene overexpression and high levels of immune infiltration abundance and immune checkpoints; Three DEGs-related gene clusters were likewise strongly linked to stromal and immunological activation. In the ICD-related prognostic model consisting of NKX3-2, CHODL, MMP1, NR0B1, and CTSV, the low-risk group patients had a better endpoint and displayed increased susceptibility to immunotherapy and chemotherapeutic drugs like 5-Fluorouracil, afatinib, bortezomib, cediratinib, lapatinib, dasatinib, gefitinib and crizotinib. Moreover, NKX3-2 amplification in HCC samples has been verified by experiments, and its disruption suppressed the proliferation and invasion of tumor cells. Conclusion: Our study highlighted the potential of the ICDRGs risk score as a prognostic indicator to aid in the accurate diagnosis and immunotherapy sensitivity of HCC.

Oncogenic PAX6 elicits CDK4/6 inhibitor resistance by epigenetically inactivating the LATS2-Hippo signaling pathway.

Intrinsic resistance to CDK4/6 inhibitors hinders their clinical utility in cancer treatment. Furthermore, the predictive markers of CDK4/6 inhibitors in gastric cancer (GC) remain incompletely described. Here, we found that PAX6 expression was negatively correlated with the response to palbociclib in vitro and in vivo in GC. We observed that the PAX6 expression level was negatively correlated with the overall survival of GC patients and further showed that PAX6 can promote GC cell proliferation and the cell cycle. The cell cycle is regulated by the interaction of cyclins with their partner serine/threonine cyclin-dependent kinases (CDKs), and the G1/S-phase transition is the main target of CDK4/6 inhibitors. Therefore, we tested whether PAX6 expression was correlated with the GC response to palbociclib. We found that PAX6 hypermethylates the promoter of LATS2 and inactivates the Hippo pathway, which upregulates cyclin D1 (CCND1) expression. This results in a suppressed response to palbociclib in GC. Furthermore, we found that the induction of the Hippo signaling pathway or treatment with a DNA methylation inhibitor could overcome PAX6-induced palbociclib resistance in GC. These findings uncover a tumor promoter function of PAX6 in GC and establish overexpressed PAX6 as a mechanism of resistance to palbociclib.

Neutrophil-to-Apolipoprotein A1 Ratio Predicted Overall Survival in Hepatocellular Carcinoma Receiving Transarterial Chemoembolization.

PURPOSE: The purpose of this study was to investigate the predictive capability of neutrophil-to-apolipoprotein A1 ratio (NAR) for predicting overall survival (OS) among patients with hepatocellular carcinoma (HCC) receiving transarterial chemoembolization (TACE). PATIENTS AND METHODS: We investigated the clinical features of 554 patients with HCC receiving TACE and assessed NAR's predictive value for OS with 222 patients (the discovery cohort) and 332 patients (the validation cohort). The association of NAR with circulation lectin-type oxidized low-density lipoprotein receptor-1-positive (LOX-1+ ) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was illustrated. RESULTS: Multivariate Cox regression revealed that lymphocyte count; Tumor, Node, Metastasis (TNM) stage; and NAR were independent prognostic factors in the discovery cohort. The validation cohort confirmed the independent prognostic value of TNM stage and NAR. Patients with low NAR (<2.7) displayed significantly increased OS in the discovery cohort (59.8 months vs. 21 months), the validation group (38.0 months vs. 23.6 months), and the total cohort (44.1 months vs. 22.0 months). A Cox proportional hazards model was used to combine Cancer of the Liver Italian Program (CLIP) score with discretized NAR. C-index illustrated that NAR-integrated CLIP score was the best model compared with NAR and CLIP score. Furthermore, NAR-CLIP presented superior predictive capacity for 10-, 20-, 30-, 40-, 50-, and 60-month survival compared with CLIP score by survival receiver-operator characteristic analysis in the discovery cohort, validation cohort, and total cohort. NAR was significantly associated with LOX-1+ PMN-MDSCs by linear regression. CONCLUSION: This study identified NAR as an independent predictor for OS among patients with HCC receiving TACE. NAR reflected circulation LOX-1+ PMN-MDSC level. IMPLICATIONS FOR PRACTICE: The present study identified neutrophil-to-apolipoprotein A1 ratio (NAR) as an independent predictor for overall survival among patients with hepatocellular carcinoma receiving transarterial chemoembolization. NAR reflected circulation level of lectin-type oxidized low-density lipoprotein receptor-1-positive polymorphonuclear myeloid-derived suppressor cells.

Intratumoral CD45+CD71+ erythroid cells induce immune tolerance and predict tumor recurrence in hepatocellular carcinoma.

CD45+CD71+ erythroid cells generated through splenic extramedullary erythropoiesis have recently been found to suppress anti-infection and tumor immunity in neonates and adults with malignances. However, their role in tumor microenvironment has not been investigated. In the present study, we found that the number of CD45+CD71+ erythroid cells was significantly elevated in hepatocellular carcinoma (HCC) tissues compared to that in paratumor region and circulation. Additionally, they were more abundant in HCC tissues compared to some immune suppressive cells as well as CD45-CD71+ erythroid cells. CD45+CD71+ erythroid cells suppressed T cells through generation of reactive oxygen species, IL-10, and TGF-ß in a paracrine and cell-cell contact manner, and their suppressive effect was stronger than that of myeloid-derived suppressor cells. The abundance of CD45+CD71+ erythroid cells in tumor tissue, as illustrated via immunofluorescence, predicted disease-free survival and overall survival, and its prognostic value was better than that of Cancer of the Liver Italian Program score. This study demonstrated that accumulation of intratumoral CD45+CD71+ erythroid cells in HCC tissues could play a superior immunosuppressive role in tumor microenvironment and may serve as a valuable biomarker to predict recurrence of HCC.

Comparison of Face-Touching Behaviors Before and During the Coronavirus Disease 2019 Pandemic.

Importance: There is insufficient evidence on the efficacy of masks in the general population for the prevention of coronavirus disease 2019 (COVID-19) in public areas. Therefore, it is imperative to investigate the association of mandatory mask-wearing policies with behaviors associated with the transmission of COVID-19. Objective: To assess the association of mask wearing with face-touching behavior among the general population in public areas. Design, Setting, and Participants: This cross-sectional study used videos recorded in public transportation stations, streets, and parks among the general population in China, Japan, South Korea, Western Europe (ie, England, France, Germany, Spain, and Italy), and the US to analyze mask-wearing and face-touching behavior in public areas. Videos before the COVID-19 pandemic were defined as those recorded from January 2018 to October 2019, and those during the COVID-19 pandemic were defined as those recorded during February 2020 to March 2020 in China, Japan, and South Korea and during March 2020 in Western Europe and the US. Individuals who clearly displayed their face and face-touching behavior were included, and those whose behaviors were influenced by filming or public events were excluded. Exposures: Mandatory mask-wearing policies enacted at various time points in China, Japan, South Korea, Western Europe, and the US. Main Outcomes and Measures: Proportion of individuals wearing masks and incidence of face touching. Results: This study included 4699 individuals before the COVID-19 pandemic and 2887 individuals during the pandemic. During the periods studied, mask wearing increased in all regions except the US, from 20 of 1745 individuals (1.1%) to 1090 of 1097 individuals (99.4%) in mainland China (P < .001), 44 of 1422 individuals (3.1%) to 346 of 893 individuals (38.7%) in Japan (P < .001), 6 of 717 individuals (0.8%) to 277 of 324 individuals (85.5% ) in South Korea (P < .001), 1 of 546 individuals (0.2%) to 6 of 379 individuals (1.6%) in Western Europe (P = .02), and 1 of 269 individuals (0.4%) to 4 of 194 individuals (2.1%) in the US (P = .17). Surgical masks were predominant in China (989 masks [89.1%]), and fabric masks were predominant in the other regions (Japan: 371 masks [95.1%]; South Korea: 240 masks [84.8%]; Western Europe: 6 masks [85.7%]; US: 5 masks [100%]). Face-touching behaviors decreased from before COVID-19 to during COVID-19 among individuals in China (72 incidences of 1745 observations [4.1%] to 12 incidences of 1097 observations [1.1%]; P < .001), South Korea (80 incidences of 717 observations [11.2%] to 7 incidences of 324 observations [2.2%]; P < .001), and Europe (62 incidences of 546 observations [11.4%] to 23 incidences of 379 observations [6.1%]; P = .01). Logistic regression found that mask wearing was associated with a reduction in face touching in China (odds ratio [OR], 3.91; 95% CI, 2.11-7.24) and South Korea (OR, 6.69; 95% CI, 2.69-16.69) and of touching the nose, mouth, and eyes (China: OR, 8.60; 95% CI, 2.65-27.86; South Korea: OR, 29.27; 95% CI, 1.79-478.22). Conclusions and Relevance: The findings of this cross-sectional study suggest that mandatory mask-wearing policies were associated with increased mask wearing during the COVID-19 pandemic. Mask wearing was associated with reduced face-touching behavior, especially touching of the eyes, nose, and mouth, which may prevent contact transmission of COVID-19 among the general population in public areas.

Importance of activated hepatic stellate cells and angiopoietin-1 in the pathogenesis of hepatocellular carcinoma.

Previous studies have determined that activated hepatic stellate cells (aHSCs) promote the progression of hepatocellular carcinoma (HCC) by increasing angiogenesis in cancerous tissues. In addition, angiopoietin 1 (Ang­1) has been reported to be involved in tumor growth and metastasis via the promotion of angiogenesis. It remains unclear whether aHSCs and Ang­1 are involved in the angiogenesis in HCC. A total of 25 HCC and tumor­adjacent tissues, and 21 normal liver tissues were used in the present study. Immunohistochemistry (IHC) was used to detect the expression of Ang­1 and α smooth muscle actin (α­SMA). The expression of CD34 was also analyzed using IHC to evaluate the microvessel density (MVD). The protein expression levels of Ang­1 were evaluated using western blot analysis. The association between aHSC, Ang­1 and angiogenesis was determined using Spearman's rank correlation coefficient. The present study determined that the expression of α­SMA, Ang­1 and MVD (CD34) was significantly higher in the HCC tissues when compared with tumor­adjacent tissues and normal liver tissues. Spearman's rank analysis identified a positive correlation between the expression of α­SMA, Ang­1 and CD34. This suggests that α­SMA­positive aHSCs promoted angiogenesis by expressing Ang­1, resulting in the proliferation and metastasis of HCC.

The deterministic chaos and random noise in turbulent jet.

A turbulent flow is usually treated as a superposition of coherent structure and incoherent turbulence. In this paper, the largest Lyapunov exponent and the random noise in the near field of round jet and plane jet are estimated with our previously proposed method of chaotic time series analysis [T. L. Yao, et al., Chaos 22, 033102 (2012)]. The results show that the largest Lyapunov exponents of the round jet and plane jet are in direct proportion to the reciprocal of the integral time scale of turbulence, which is in accordance with the results of the dimensional analysis, and the proportionality coefficients are equal. In addition, the random noise of the round jet and plane jet has the same linear relation with the Kolmogorov velocity scale of turbulence. As a result, the random noise may well be from the incoherent disturbance in turbulence, and the coherent structure in turbulence may well follow the rule of chaotic motion.

Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure.

AIM: To study the clinical efficacy of traditional Chinese medicine (TCM) intervention "tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment" ("TTK") for treating liver failure due to chronic hepatitis B. METHODS: We designed the study as a randomized controlled clinical trial. Registration number of Chinese Clinical Trial Registry is ChiCTR-TRC-12002961. A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study. Participants were randomly assigned to the following three groups: (1) a modern medicine control group (MMC group, 36 patients); (2) a "tonifying qi and detoxification" ("TQD") group (72 patients); and (3) a "tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment" ("TTK") group (36 patients). Patients in the MMC group received general internal medicine treatment; patients in the "TQD" group were given a TCM formula "tonifying qi and detoxification" and general internal medicine treatment; patients in the "TTK" group were given a TCM formula of "TTK" and general internal medicine treatment. All participants were treated for 8 wk and then followed at 48 wk following their final treatment. The primary efficacy end point was the patient fatality rate in each group. Measurements of various virological and biochemical indicators served as secondary endpoints. The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups. RESULTS: At the 48-wk post-treatment time point, the patient fatality rates in the MMC, "TQD", and "TTK" groups were 51.61%, 35.38%, and 16.67%, respectively, and the differences between groups were statistically significant (P < 0.05). However, there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups (P > 0.05). Patients in the "TTK" group had significantly higher levels of serum total bilirubin compared to MMC subjects (339.40 µmol/L ± 270.09 µmol/L vs 176.13 µmol/L ± 185.70 µmol/L, P = 0.014). Serum albumin levels were significantly increased in both the "TQD" group and "TTK" group as compared with the MMC group (31.30 g/L ± 4.77 g/L, 30.72 g/L ± 2.89 g/L vs 28.57 g/L ± 4.56 g/L, P < 0.05). There were no significant differences in levels of alanine transaminase among the three groups (P > 0.05). Safety data showed that there was one case of stomachache in the "TQD" group and one case of gastrointestinal side effect in the "TTK" group. CONCLUSION: Treatment with "TTK" improved the survival rates of patients with liver failure due to chronic hepatitis B. Additionally, liver tissue was regenerated and liver function was restored.

Estimating the largest Lyapunov exponent and noise level from chaotic time series.

A novel method for estimating simultaneously the largest Lyapunov exponent (LLE) and noise level (NL) from a noisy chaotic time series is presented in this paper. We research the influence of noise on the average distance of different pairs of points in an embedding phase space and provide a rescaled formula for calculating the LLE when the time series is contaminated with noise. Our algorithm is proposed based on this formula and the invariant of the LLE in different dimensional embedding phase spaces. With numerical simulation, we find that the proposed method provides a reasonable estimate of the LLE and NL when the NL is less than 10% of the signal content. The comparison with Kantz algorithm shows that our method gives more accurate results of the LLE for the noisy time series. Furthermore, our method is not sensitive to the distribution of the noise.

Synthesis of a novel series of diphenolic chromone derivatives as inhibitors of NO production in LPS-activated RAW264.7 macrophages.

A novel series of diphenolic chromone derivatives were synthesized and their inhibitory activity on nitric oxide (NO) production and cytotoxicity were evaluated using LPS-activated murine macrophages RAW264.7 assay and MTT method, respectively. Among these compounds, (5,7-dihydroxy-4-oxo-4H-chromen-3-yl) methyl esters (6b, 6c, 6f, 6g, and 6h) showed quite potent inhibitory activities with IC(50) values of 2.20, 3.48, 0.35, 0.80, and 0.61microM, respectively. The MTT results showed that all of the active compounds exhibited no cytotoxicity at the effective concentrations. The preliminary mechanism of the most potent compounds (6b, 6c, 6f, 6g, and 6h) was further examined based on the RT-PCR results and the compounds 6f, 6g, and 6h inhibited NO production by suppressing the expression of iNOS mRNA in a dose dependent manner. Furthermore, a computational analysis of physicochemical parameters revealed that the most of the compounds possessed drug-like properties.

Synthesis and evaluation of a novel series of quinoline derivatives with immunosuppressive activity.

A series of quinoline derivatives were synthesized and their immunosuppressive activity and cytotoxicity were evaluated with a T-cell functional assay and MTT method, respectively. Most of 5,7-dimethoxyquinolin-4-yl ortho-substituted benzoate derivatives (18, 22, 24, 28, 31 and 37) showed a quite stronger inhibitory activity compared to other analogs. Among the synthesized compounds, 5,7-dimethoxyquinolin-4-yl 2,6-dichlorobenzoate (22) and 5,7-dimethoxyquinolin-4-yl 4-methylbenzenesulfonate (40) exhibited a potent inhibitory activity without significant cytotoxicity at 10 microM concentration. The preliminary mechanism of the active compounds 22 and 40 was further clarified based on the fluorescence activated cell sorter (FACS) assay, and the compounds exerted immunosuppressive activity via inhibiting the T cell activation in a dose dependent manner.

A novel synthetic oleanolic acid derivative with amino acid conjugate suppresses tumour growth by inducing cell cycle arrest.

Oleanolic acid (3beta-hydroxy-olean-12-en-28-oic acid; OA) has a wide variety of bioactivities and is used for medicinal purposes in many Asian countries. Various derivatives of OA have been synthesized in attempts to improve the potency. Here we describe the anti-tumour activity of a novel OA derivative, N-[(3beta)-3-(acetyloxy)-28-oxoolean-12-en-28-yl]-glycine methyl ester (AOA-GMe). AOAGMe was a more potent inhibitor of the growth of B16 melanoma cells than its parent compound OA, both in-vitro and in-vivo. AOA-GMe also exhibited dose-dependent inhibition of human K562 leukaemia cells, but had almost no toxicity in normal human peripheral blood mononuclear cells. AOA-GMe induced cell cycle arrest in G0/G1 and blocked G1-S transition, which correlated well with marked decreases in levels of cyclin D, cyclin-dependent kinase CDK4 and phosphorylated retinoblastoma protein, and increases in the cyclin-dependent kinase inhibitor p15. OA did not show such activities. These results suggest that AOA-GMe may induce growth arrest in tumour cells through regulation of proteins involved in the cell cycle.

Inversion formula of multifractal energy dissipation in three-dimensional fully developed turbulence.

The concept of inverse statistics in turbulence has attracted much attention in recent years. It is argued that the scaling exponents of the direct structure functions and the inverse structure functions satisfy an inversion formula. This proposition has already been verified by numerical data using the shell model. However, no direct evidence was reported for experimental three-dimensional turbulence. We propose to test the inversion formula using experimental data of three-dimensional fully developed turbulence by considering the energy dissipation rates instead of the usual efforts on the structure functions. The moments of the exit distances are shown to exhibit nice multifractality. The inversion formula between the direct and inverse exponents is then verified.