Inhibition of human adenovirus replication by TRIM35-mediated degradation of E1A.

Human adenovirus (HAdV) is ubiquitous in the human population, constituting a significant burden of global respiratory diseases. Children and individuals with low immunity are at risk of developing severe infections without approved antiviral treatment for HAdV. Our study demonstrated that TRIM35 inhibited HAdV-C5 early gene transcription, early protein expression, genome replication, and infectious virus progeny production. Furthermore, TRIM35 was found to inhibit HAdV replication by attenuating E1A expression. Mechanistically, TRIM35 interacts with and degrades E1A by promoting its K48-linked ubiquitination. Additionally, K253 and K285 are the key sites necessary for TRIM35 degradation. Moreover, an oncolytic adenovirus carrying shTRIM35 was constructed and observed to exhibit improved oncolysis in vivo, providing new ideas for clinical tumor treatment. Our results expand the broad antiviral activity of TRIM35 and mechanically support its application as a HAdV replication inhibitor. IMPORTANCE E1A is an essential human adenovirus (HAdV) protein responsible for the early replication of adenovirus while interacting with multiple host proteins. Understanding the interaction between HAdV E1A and TRIM35 helps identify effective antiviral therapeutic targets. The viral E1A protein is a crucial activator and regulator of viral transcription during the early infection stages. We first reported that TRIM35 interacts with E1A to resist adenovirus infection. Our study demonstrated that TRIM35 targets E1A to resist adenovirus, indicating the applicability of targeting virus-dependent host factors as a suitable antiviral strategy.

Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio as predictive markers in hepatoblastoma.

Background: The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been presented to be a prognostic indicator in several cancers. We were supposed to evaluate the prognostic role of such inflammatory markers in hepatoblastoma (HB). Methods: Total of 101 children, diagnosed with hepatoblastoma between January 2010 and January 2018, were enrolled before treatment in the study. The clinicopathological parameters, and outcomes were collected through laboratory analyses and patient follow-up. The association between NLR, PLR, and clinicopathological characters were analyzed with Wilcoxon test, Chi-Squared test, Kaplan-Meier, Log-rank and Cox regression analyses. Results: NLR and PLR were significantly elevated in HB patients (P < 0.001), and related to age (P < 0.001), risk stratification system (P < 0.001), and pretreatment extent of disease (P < 0.0001). NLR was significantly related to alpha-fetoprotein (P = 0.034) and lactate dehydrogenase (P = 0.026). The 3-year overall survival (OS) and event-free survival (EFS) were poor in the high-NLR group (OS: 44.3% vs. 90.3%, P < 0.0001, EFS: 38.6% vs. 80.6%, P = 0.0001). The 3-year OS and EFS were poor in the high-PLR group (OS: 49.1% vs. 68.8%, P = 0.016, EFS: 39.6% vs. 64.6%, P = 0.0117). The multivariate analysis suggested that NLR (HR: 11.359, 95% CI: 1.218-105.947; P = 0.033) and risk stratification (HR: 44.905, 95% CI: 2.458-820.36; P = 0.01), were independent predictors of OS. Conclusion: Our research showed that elevated NLR and PLR were the poor prognostic factors in HB patients before treatment. The NLR was an independent prognostic factor for OS.

Preparation and In Vitro and In Vivo Evaluation of a Testosterone Film Forming Gel for the Treatment of Hypoactive Sexual Desire Disorder in Women.

Hypoactive sexual desire disorder (HSDD) is one of the most common sexual complaints in women. Currently, there is an unmet need for a drug treatment for this disorder. The purpose of this study was to develop a testosterone (TS) film forming gel used for women to treat HSDD by measuring the tackiness, peel adhesion force, tensile strength, and elasticity of the formulation. Diethylene glycol monoethyl ether (Transcutol P), an efficient penetration enhancer, was added to the optimized formulation and the transdermal permeation characteristics in vitro were studied using Franz-diffusion cells. The quantitative determination of TS was performed by high-performance liquid chromatography (HPLC). After 24 h, Transcutol P at 3% had the largest cumulative amount of drug and enhancement ratio of TS of 75.14 µg/cm2 and 2.82, respectively. After the screening of film forming polymers and penetration enhancers, the optimal formulation was as follows: glycerol (1%, w/w); 12.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution (0.5%, w/w); 2.5% Carbomer ethanol solution (0.5%, w/w); Transcutol P ethanol solution (3%, w/w) containing 0.5% TS; and 8% Poly vinyl alcohol (PVA) aqueous solution (30%, w/w). The optimized film forming gel had good uniformity and the release of TS in vitro was close to 100% within 24 h. In vivo studies showed the formulations had optimal area under blood drug concentration curve values in the order of 3% Transcutol P > 1% Transcutol P > 5% Transcutol P > control preparation. The formulation with 3% Transcutol P provided the highest permeation effect both in vitro and in vivo. The safety of this formulation was further evaluated with a skin irritation test. It could effectively improve the rabbit skin irritation observed with a marketed transdermal patch Androderm®. The present study provides a promising approach for the development of a novel TS film forming gel for the treatment of HSDD in women.

L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma.

OBJECTIVE: L1 cell adhesion molecule (L1CAM) exhibits oncogenic activity in tumors. However, the link between L1CAM and the tumor microenvironment remains poorly understood in patients with esophageal squamous cell carcinoma (ESCC). In this study, we investigated how L1CAM expression in ESCC affects the oncogenic characteristics of tumor cells and the tumor microenvironment. METHODS: Human ESCC samples were collected, and the mRNA and protein levels of L1CAM were examined by real-time PCR and immunohistochemistry. Overexpression and knockdown gene expression assays were used for mechanistic studies. The cell proliferation and cell cycle were measured with CCK-8 assays and flow cytometry. Cell migration and invasion ability were measured with Transwell assays. Multiplex bead-based assays were performed to identity the factors downstream of L1CAM. Xenograft studies were performed in nude mice to evaluate the effects of L1CAM on tumor growth and regulatory T cell (Treg) recruitment. RESULTS: L1CAM expression was significantly elevated in ESCC tissues (P < 0.001) and correlated with poorer prognosis (P < 0.05). Ablation of L1CAM in ESCC cells inhibited tumor growth and migration, and increased tumor cell apoptosis (P < 0.05). In the tumor microenvironment, L1CAM expression correlated with Treg infiltration in ESCC by affecting CCL22 secretion. Mechanistically, L1CAM facilitated CCL22 expression by activating the PI3K/Akt/NF-κB signaling pathway. Furthermore, CCL22 promoted Treg recruitment to the tumor site; the Tregs then secreted TGF-ß, which in turn promoted L1CAM expression via Smad2/3 in a positive feedback loop. CONCLUSIONS: Our findings provide new insight into the mechanism of immune evasion mediated by L1CAM, suggesting that targeting L1CAM-CCL22-TGF-ß crosstalk between tumor cells and Tregs may offer a unique means to improve treatment of patients with ESCC.

Evaluation of the diagnostic value of circulating tumor cells with CytoSorter® CTC capture system in patients with breast cancer.

PURPOSE: In this study, we aimed to investigate the viability of utilizing CytoSorter® system to detect circulating tumor cells (CTCs) and to evaluate the diagnostic value of CTCs in breast cancer (BC). METHODS: A total of 366 females patients suspected of having BC and 30 healthy female volunteers were enrolled in this study. CTCs were enriched by CytoSorter® , a microfluidic-based CTCs capturing platform. CTC detection was performed before operation or biopsy. Based on the biopsy results, patients were divided into two groups, namely patients with BC and patients with benign breast diseases (BBD). Patients with BBD and healthy volunteers were serving as controls. The correlation between CTC enumeration and patients' clinicopathological characteristics was evaluated. The receiver operating characteristic (ROC) curve was plotted to assess the diagnostic potency of CytoSorter® system in BC. RESULTS: Based on the biopsy results, 130 BC patients at different cancer stages and 236 patients with BBD were enrolled in the study. Seven subjects were dropped out from the study. CTCs were detected in 109 of 128 BC patients, in one of 29 healthy volunteers, and in 37 of 232 patients with BBD. Maximum CTC counts detected in BC patients, healthy volunteers, and patients with BBD were 8, 1, and 4, respectively. Statistical analysis showed CTCs could be used to distinguish BC patients from healthy volunteers and patients with BBD (P < .0001). Circulating tumor cells were statistically associated with patients' cancer stage (P = .0126), tumor size (tumor node metastasis [TNM] T stage, P = .0253), cancer type (invasive vs noninvasive, P = .0141), and lymph node metastasis (P = .0436). More CTCs were found in patients at advanced cancer stage or TNM T stage and in patients with invasive tumor or lymph node metastasis. Furthermore, CTC detection rates in BC patients at Tis and T1-4 stages were 50%, 81.67%, 91.07%, 100%, and 100%, respectively. When the CTC cut-off value was set to 2, the ROC curve gave an area under the curve (AUC) of 0.86 with a specificity and sensitivity of 95.4% and 76.56%, respectively. Taken together, CTCs could be used as a diagnostic aid in assistance of cancer screening and staging. CONCLUSION: Circulating tumor cells were successfully isolated in BC patients using CytoSorter® system. CTCs can be used to differentiate BC patients from the patients with BBD or healthy volunteers, and as a diagnostic aid for early cancer diagnosis and cancer staging.

[Effect and mechanism of total flavonoids of Lichi Semen on CCl_4-induced liver fibrosis in rats, and prediction of Q-marker].

This paper was to investigate the effect of total flavonoids of Lichi Semen(TFL) on carbon tetrachloride(CCl_4)-induced liver fibrosis in rats, analyze and predict its mechanism of action and potential quality markers(Q-marker). Firstly, male SD rats were taken and injected subcutaneously with a 40% CCl_4-vegetable oil solution twice a week for 8 consecutive weeks to establish a rat model of liver fibrosis. The rats with liver fibrosis were randomly divided into model group, silybin group(43.19 mg·kg~(-1)), Fuzheng Huayu Capsules group(462.75 mg·kg~(-1)), and TFL groups(100 mg·kg~(-1) and 25 mg·kg~(-1)), with normal rats as a blank group, 10 rats in each group. Except for the blank group, the rats in the other groups were subcutaneously injected with 40% CCl_4-vegetable oil solution of a maintenance dose, once a week. The rats in various treatment groups received corresponding doses of drugs, while the rats in the blank group and model group received the same volume of normal saline once a day for 4 weeks. At the end of the experiment, blood was collected from the abdominal aorta and the liver tissues were collected. The levels of total bilirubin(TBiL), direct bilirubin(DBiL), indirect bilirubin(IBiL), alanine aminotransferase(ALT), and aspartate aminotransferase(AST) in serum were detected by using an automatic biochemical detector. Masson staining was used to observe the histopathological changes of rat liver. Then, the chemical compositions of TFL were collected, and the action targets of these chemical compositions were predicted through SWISS database and reverse molecular docking server(DRAR-CPI). After screening of disease targets of liver fibrosis by Gene Cards database, the protein-protein interaction was analyzed with use of STRING database, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) enrich analysis were also carried out. Moreover, an iTRAQ proteomics technology was used to determine protein expression in liver tissues of rats in TFL, model and blank groups to verify the targets. Furthermore, Cytoscape software was used to establish and visualize the network of chemical components, targets and pathways, and predict the potential Q-marker of TFL. The results showed that the levels of TBiL, DBiL, IBiL, ALT, and AST in the model group were significantly higher than those in the blank normal group(P<0.05), and the above levels in the treatment groups were lower than those in the model group, but with no significant differences. Masson staining showed that the liver damage and the degree of fibrosis were severe in the model group, and were relieved to different degrees in the treatment groups. Then, 74 chemical components were screened, which could act on 865 targets such as EGFR and SRC, participating in the regulation of cancer pathways, PI3 K-Akt signaling pathway, HIF-1 signaling pathway and other signaling pathways closely related to liver fibrosis. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin showed the highest correlation with liver fibrosis-related targets and pathways. Proteomics results showed that a total of 18 proteins among the 45 proteins predicted by internet pharmacology were identified, among which 6 proteins were significantly expressed, including 5 up-regulated proteins and 1 down-regulated protein. The protein expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1 was significantly returned to a normal state in the TFL treatment groups. In conclusion, TFL may demonstrate the anti-hepatic fibrosis and potential hepatoprotective effects by regulating the expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1, which may be associated with the regulation of multiple signaling pathways related to liver fibrosis such as PI3 K-Akt pathway. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin could be regarded as potential Q-markers of TFL for quality control.

Cytokine induced killer cell-based immunotherapies in patients with different stages of renal cell carcinoma.

Cytokine induced killer (CIK) cell-based treatments have shown antitumor activity against renal cell carcinoma (RCC) in vitro and in vivo. But the therapeutic options and benefits of various CIK cells were unknown for different stages of RCC. In this random clinical trial, the 3-year disease free survival (DFS) of operable RCC patients treated with autologous tumor lysate-pulsed dendritic cells co-cultured with cytokine induced killer (Ag-DC-CIK) was 96.7% compared with 57.7% in the control group. Ag-DC-CIK immunotherapy decreased the risk of post-operative disease progression and relapse (P = 0.0418). In inoperable RCC patients treated with CIK, the 3-year overall survival (OS) and progression-free survival (PFS) were significantly longer than the control group (P = 0.0116 and P = 0.0212). The CD4(+)/CD8(+) T cell ratio in peripheral blood increased after the last cell infusion in the CIK treatment group, and especially further increased in the Ag-DC-CIK treatment group (P = 0.002). No severe toxicity was observed after infusion of CIK cells. Therefore, tumor antigen-sensitized Ag-DC-CIK cells might be more efficient and personalized for the patients with tumor resection, and CIK cells could improve the prognosis for those inoperable patients. According to the stages of RCC patients, different CIK cell-based immunotherapies would help to achieve more beneficial effects.