HPV-16 E6 mutation and viral integration related host DNA methylation implicate the development and progression of cervical cancer.

BACKGROUND: HPV-16 infection and viral-host integration are the most important risk factors for cervical cancer (CC). The aim of this study is to develop a new molecular strategy integrated both the viral and host genome variations identifying and monitoring CC. METHOD: A total of 312 methylation and 538 RNA-seq datasets were collected from public databases to identify differentially methylated and expressed genes. HPV associated virus integration sites (VISs) were analysed using the ViMIC database. From September 2020 to August 2021, the 70 HPV-16 positive cases retrospectively collected from multi-centre cohorts were subjected to HPV-16 E6 deep sequencing and PCR-based host gene (ASTN1, DLX1, ITGA4, RXFP3, SOX17, ZNF671) methylation detection. RNAseq and expression validation (NNF671) were performed in C-33A cell line harbouring HPV D32E. Lasso and logistic regression algorithm were used to construct the CC diagnostic model. RESULTS: A positive correlation was observed between the average methylation level of CC patients and their pathological features including tumour stage (p = 0.0077) and HPV subtype (p < 0.001). ZNF671 was identified as a CC-specific methylation marker, with an impressive 93% sensitivity. Both HPV-16 D32E mutation and integration of HPV-16 down-regulated the ZNF671 expression. Finally, a CC diagnostic nomogram was developed by integrating ZNF671 methylation level and HPV E6 mutation feature, yielding an exceptional AUC of 0.997 (95% CI: 0.934-1.000). CONCLUSIONS: Our study demonstrated HPV viral mutations are closely related to host gene epigenetic alterations in CC. Integration of the viral and host genetic information might be a new promising strategy for CC screening.

Deep Sequencing of HPV16 E6 Region Reveals Unique Mutation Pattern of HPV16 and Predicts Cervical Cancer.

The genetic diversity of human papillomavirus (HPV) 16 within cervical cells and tissue is usually associated with persistent virus infection and precancerous lesions. To explore the HPV16 mutation patterns contributing to the cervical cancer (CC) progression, a total of 199 DNA samples from HPV16-positive cervical specimens were collected and divided into high-grade squamous intraepithelial lesion (HSIL) and the non-HSIL(NHSIL) groups. The HPV16 E6 region (nt 7125-7566) was sequenced using next-generation sequencing. Based on HPV16 E6 amino acid mutation features selected by Lasso algorithm, four machine learning approaches were used to establish HSIL prediction models. The receiver operating characteristic was used to evaluate the model performance in both training and validation cohorts. Western blot was used to detect the degradation of p53 by the E6 variants. Based on the 13 significant mutation features, the logistic regression (LR) model demonstrated the best predictive performance in the training cohort (AUC = 0.944, 95% CI: 0.913-0.976), and also achieved a high discriminative ability in the independent validation cohort (AUC = 0.802, 95% CI: 0.601-1.000). Among these features, the E6 D32E and H85Y variants have higher ability to degrade p53 compared to the E6 wildtype (P < 0.05). In conclusion, our study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting HSIL. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of CC. IMPORTANCE The study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting the high-grade squamous intraepithelial lesion and can reduce even more unneeded colposcopies without a loss of sensitivity to detect cervical cancer. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of cervical cancer.

Quantitative detections of TP53 gene mutations improve the diagnosis and prognostic prediction of biliary tract cancers using droplet digital PCR.

OBJECTIVE: Biliary tract cancer (BTC) is a rare malignancy and lack of effective diagnostic and prognostic marker. Here, we aimed to investigate the clinical implication of TP53 mutation detection in BTC using droplet digital PCR (ddPCR). METHODS: TP53 gene (loci p.R175H, p.R248Q, p.R248W, and p.R273H) mutation frequencies of 45 pairs of tumor tissues (TTs) and adjacent normal tissues (ANTTs) were analyzed, respectively, using ddPCR. Meanwhile, the same detections were conducted in plasma cell-free DNA (cfNDA) of 156 subjects including BTC, disease control (DC), and healthy controls (HC). The logistic regression algorithm was established to identify BTC. The correlations between mutations and clinicopathological features as well as the effects of TP53 mutation frequency on BTC prognosis were assessed. RESULTS: The higher mutation of p.R175H was found in TTs compared with ANTT (p = 0.006). The mutation at p.R273H in cfDNA was also higher in BTC when compared with DC and HC (p < 0.05). The logistic algorithms combining p.R273H mutation demonstrated the higher diagnostic efficacy trend than carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) in identifying BTC from DC (the area under the curves of the algorithm: 0.845, 95% CI:0.775-0.914). The median overall survival (OS) and progression-free survival (PFS) were significantly shorter when the BTC patients harboring the p.R273H mutation (OS: p = 0.032; PFS: p = 0.046). CONCLUSION: This study revealed for the first time that the quantitative TP53 mutations using the ddPCR might serve as a potential genetic biomarker for BTC diagnosis and prognosis assessment.

Blood tests predict the therapeutic prognosis of anti-PD-1 in advanced biliary tract cancer.

Predictive prognostic markers for immunotherapy are crucial and desperately required for clinical precise medicine. This retrospective study aimed to assess the efficacy of anti-PD-1 (programmed cell death protein 1) treatment and find the therapeutic prognostic biomarkers in advanced biliary tract cancer (BTC). A total of 60 patients of advanced BTC who received at least one dose of anti-PD-1 therapy between June 2016 and October 2019 were recruited and followed up till April 2020. Systemic immune-inflammation index (SII) and neutrophils-to-lymphocytes ration (NLR) were obtained from the routine circulating hematologic analysis before treatment. Serum 45-Plex Panel cytokines were detected using multiplexed bead immunoassays. Logistic regression nomogram was used to construct the algorithm model for prognosis prediction. Of the 60 patients, the overall benefit rate (OBR) was 38.3%, the median progression free survival (PFS), and overall survival (OS) were 4.0 mo (95% confidence interval [CI]: 2.28-5.72) and 13.0 mo (95% CI: 8.05-17.95), respectively. High levels of SII (≥720), NLR (≥4.3) and cytokine IFN-inducible protein-10 (IP-10; ≥45 pg/ml) indicated worse OS. Those with high SII (≥720) and high IP-10 (≥45 pg/ml) also had shorter PFS. The nomogram algorithm combining above three independent factors (SII, IP-10, and macrophage inflammatory protein-1ß) had better efficacy in predicting OBR. Our study offers a simple, affordable, and noninvasive method to help physicians predict therapeutic response in BTC patients receiving anti-PD-1 antibody treatment.

Single and 14-day repeated dose inhalation toxicity studies of hexabromocyclododecane in rats.

Limited toxicological information is available for hexabromocyclododecane (HBCD),a widely used additive brominated flame retardant. Inhalation is a major route of human exposure to HBCD. The aim of this study was to determine the acute inhalation toxicity and potential subchronic inhalation toxicity of HBCD in Sprague-Dawley rats exposed to HBCD only through inhalation. The acute inhalation toxicity of HBCD was determined using the limit test method on five male and five female Sprague-Dawley rats at a HBCD concentration of 5000 mg/m(3). Repeated-dose toxicity tests were also performed, with 20 males and 20 females randomly assigned to four experimental groups (five rats of each sex in each group). There were three treatment groups (exposed to HBCD concentrations of 125,500, and 2000 mg/m(3)) and a blank control group (exposed to fresh air). In the acute inhalation toxicity study, no significant clinical signs were observed either immediately after exposure or during the recovery period. Gross pathology examination revealed no evidence of organ-specific toxicity in any rat. The inhalation LC50(4 h) for HBCD was higher than 5312 ± 278 mg/m3 for both males and females. In the repeated dose inhalation study, daily head/nose-only exposure to HBCD at 132 ± 8.8, 545.8 ± 35.3, and 2166.0 ± 235.9 mg/m(3) for 14 days caused no adverse effects. No treatment-related clinical signs were observed at any of the test doses. The NOAEL for 14-day repeated dose inhalation toxicity study of HBCD is 2000 mg/m(3).

Effects of Nano-CeO2 with Different Nanocrystal Morphologies on Cytotoxicity in HepG2 Cells.

Cerium oxide nanoparticles (nano-CeO2) have been reported to cause damage and apoptosis in human primary hepatocytes. Here, we compared the toxicity of three types of nano-CeO2 with different nanocrystal morphologies (cube-, octahedron-, and rod-like crystals) in human hepatocellular carcinoma cells (HepG2). The cells were treated with the nano-CeO2 at various concentrations (6.25, 12.5, 25, 50, 100 µg/mL). The crystal structure, size and morphology of nano-CeO2 were investigated by X-ray diffractometry and transmission electron microscopy. The specific surface area was detected using the Brunauer, Emmet and Teller method. The cellular morphological and internal structure were observed by microscopy; apoptotic alterations were measured using flow cytometry; nuclear DNA, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and glutathione (GSH) in HepG2 cells were measured using high content screening technology. The scavenging ability of hydroxyl free radicals and the redox properties of the nano-CeO2 were measured by square-wave voltammetry and temperature-programmed-reduction methods. All three types of nano-CeO2 entered the HepG2 cells, localized in the lysosome and cytoplasm, altered cellular shape, and caused cytotoxicity. The nano-CeO2 with smaller specific surface areas induced more apoptosis, caused an increase in MMP, ROS and GSH, and lowered the cell's ability to scavenge hydroxyl free radicals and antioxidants. In this work, our data demonstrated that compared with cube-like and octahedron-like nano-CeO2, the rod-like nano-CeO2 has lowest toxicity to HepG2 cells owing to its larger specific surface areas.

Optimal method to stimulate cytokine production and its use in immunotoxicity assessment.

Activation of lymphocytes can effectively produce a large amount of cytokines. The types of cytokines produced may depend on stimulating reagents and treatments. To find an optimal method to stimulate cytokine production and evaluate its effect on immunotoxicity assessments, the authors analyzed production of IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, GM-CSF, RANTES and TGF-ß in undiluted rat whole blood culture (incubation for 0, 2, 4, 6, 8 or 10 h) with different concentrations of PMA/ionomycin, PHA, Con A, LPS and PWM. We also evaluated the effects of cyclosporin A and azathioprine on cytokine production. The results revealed a rapid increase of IL-2, IFN-γ, TNF-α, RANTES and TGF-ß secretion within 6 h after stimulation with 25 ng/mL PMA and 1 µg/mL ionomycin. The inhibition of these cytokine profiles reflected the effects of immunosuppressants on the immune system. Therefore, the results of this is study recommend the detection of cytokine profiles in undiluted whole blood stimulated 6 h with 25 ng/mL PMA and 1 µg/mL ionomycin as a powerful immunotoxicity assessment method.

Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways.

BACKGROUND: Lanthanide cerium oxide (CeO2) nanoparticles have extensive applications in industrial fields, and concerns regarding their potential toxicity in humans and their environmental impact have increased. We investigated the underlying molecular mechanisms by which CeO2 nanoparticles induce toxicity in human hepatoma SMMC-7721 cells. RESULTS: Our results demonstrated that CeO2 nanoparticles reduced viability, caused dramatic morphological damage, and induced apoptosis in SMMC-7721 cells. CeO2 nanoparticles significantly increased the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and significantly reduced the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT). The phosphorylation levels of ERK1/2, JNK and p38 MAPK were significantly elevated after treatment with CeO2 nanoparticles. Pretreatment with the antioxidant N-acetyl-cysteine (NAC): reduced the induction of ROS and MDA by CeO2 nanoparticles; recovered the activity of SOD, GSH-px and CAT; reduced the phosphorylation levels of ERK1/2, JNK and p38; and attenuated CeO2 nanoparticles-induced damage and apoptosis in SMMC-7721 cells. CONCLUSIONS: Our data demonstrated that CeO2 nanoparticles induced damage and apoptosis in human SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways.