N-nitrosodimethylamine exposure to zebrafish embryos/larvae causes cardiac and spinal developmental toxicity.

N-nitrosodimethylamine (NDMA), one of the new nitrogen-containing disinfection by-products, is potentially cytotoxic, genotoxic, and carcinogenic. Its potential toxicological effects have attracted a wide range of attention, but the mechanism is still not sufficiently understood. To better understand the toxicological mechanisms of NDMA, zebrafish embryos were exposed to NDMA from 3 h post-fertilization (hpf) to 120hpf. Mortality and malformation were significantly increased, and hatching rate, heart rate, and swimming behavior were decreased in the exposure groups. The result indicated that NDMA exposure causes cardiac and spinal developmental toxicity. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, and bcl-2 were significantly affected by NDMA exposure. Moreover, the genes associated with spinal and cardiac development (myh6, myh7, nkx2.5, eph, bmp2b, bmp4, bmp9, run2a, and run2b) were significantly downregulated after treatment with NDMA. Wnt and TGF-ß signaling pathways, crucial for the development of diverse tissues and organs in the embryo and the establishment of the larval spine, were also significantly disturbed by NDMA treatment. In summary, the disinfection by-product, NDMA, exhibits spinal and cardiac developmental toxicity in zebrafish embryos, providing helpful information for comprehensive analyses and a better understanding the mechanism of its toxicity.

Potentilla parvifolia strongly influenced soil microbial community and environmental effect along an altitudinal gradient in central Qilian Mountains in western China.

The Qilian Mountains (QLMs) form an important ecological security barrier in western China and a priority area for biodiversity conservation. Potentilla parvifolia is a widespread species in the mid-high altitudes of the QLMs and has continuously migrated to higher altitudes in recent years. Understanding the effects of P. parvifolia on microbial community characteristics is important for exploring future changes in soil biogeochemical processes in the QLMs. This study found that P. parvifolia has profound effects on the community structure and ecological functions of soil microorganisms. The stability and complexity of the root zone microbial co-occurrence network were significantly higher than those of bare soils. There was a distinct altitudinal gradient in the effect of P. parvifolia on soil microbial community characteristics. At an elevation of 3204 m, P. parvifolia promoted the accumulation of carbon, nitrogen, and phosphorus and increased sucrase activity and soil C/N while significantly improving the community richness index of fungi (p < .05) compared with that of bacteria and the relative abundance of Ascomycota. The alpha diversity of fungi in the root zone soil of P. parvifolia was also significantly increased at 3550 m altitude. Furthermore, the community similarity distance matrix of fungi showed an evident separation at 3204 m. However, at an altitude of 3750 m, P. parvifolia mainly affected the bacterial community. Potentilla parvifolia increased the bacterial community richness. This is in agreement with the findings based on the functional prediction that P. parvifolia favors the growth and enrichment of denitrifying communities at 3550 and 3750 m. The results provide a scientific basis for predicting the evolutionary trends of the effects of P. parvifolia on soil microbial communities and functions and have important implications for ecological governance in the QLMs.

Tumor-derived proliferative CTCs and CTC clusters predict aggressiveness and early recurrence in hepatocellular carcinoma patients.

BACKGROUND: Circulating tumor cells (CTCs), an indispensable liquid biopsy classifier, can provide extra information for the diagnosis and prognosis of hepatocellular carcinoma (HCC). METHODS: We systematically analyzed the peripheral blood of preoperative HCC patients (n = 270) for CTC number, Ki67 index reflecting the proliferative CTC percentage (PCP), and CTC clusters correlated with the characteristics of malignant HCC tumors. RESULTS: Driver gene mutations were found with high levels of consistency between CTCs and primary tumors (n = 73). CTC number and PCP were associated with tumor size, microvascular invasion (MVI), presence or absence of multiple tumors, and thrombosis significantly. CTC number and PCP robustly separated patients with and without relapse, with a sensitivity of 88.89%/81.48% and a specificity of 72.73%/94.81% in the training set (n = 104) and corresponding values of 80.00%/86.67% and 78.38%/89.19% in the validation set (n = 52), showing a better performance than that associated with the alpha-fetoprotein (AFP) level. CTC number, PCP, CTC clusters, and MVI were independent significant risk factors for HCC recurrence (P = 0.0375, P < 0.0001, P = 0.0017, and P = 0.0157). A nomogram model based on these risk factors showed a considerable prediction ability for HCC recurrence (area under the curve = 0.947). PCP (training: log-rank P < 0.0001; hazard ratio [HR] 30.13, 95% confidence interval [CI] = 11.12-81.62; validation: log-rank P < 0.0001; HR 25.73, 95% CI = 5.28-106.60) and CTC clusters (training: log-rank P < 0.0001; HR 17.34, 95% CI = 7.46-40.30; validation: log-rank P < 0.0001; HR 9.92, 95% CI = 2.55-38.58) were more significantly correlated with worse recurrence-free survival than CTC number (training: log-rank P < 0.0001; HR 14.93, 95% CI = 4.48-49.78; validation: log-rank P = 0.0007; HR 9.03, 95% CI = 2.53-32.24). CONCLUSION: PCP and CTC clusters may predict HCC recurrence and improve the performance of the serum biomarker AFP.

Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses.

BACKGROUND: Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. METHODS: Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. RESULTS: The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. CONCLUSIONS: Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

Telomere Maintenance Associated Mutations in the Genetic Landscape of Gynecological Mucosal Melanoma.

PURPOSE: Gynecological melanomas (GMs) are rare tumors with a poor prognosis. Here, we performed exome sequencing to generate the mutational landscape of GMs. METHODS: Next-generation sequencing was carried out on mucosal melanoma samples (n = 35) obtained from gynecological sites. The alternative telomere lengthening (ALT) phenotype was verified by fluorescence in situ hybridization and the C-circle assay. Immunohistochemistry was performed to detect ATRX protein. Copy number variations in TERT were detected by droplet digital polymerase chain reaction. RESULTS: In the 58 formalin-fixed paraffin-embedded samples, we identified 33 (56.9%) ALT-positive cases, with 23 showing loss of ATRX protein. TERT promoter mutation was not detected in GMs (n = 40), but copy number variations in the TERT region were observed in 20% (7/35) of the samples. TERT amplification was mutually exclusive with ALT (P < 0.05). Kaplan-Meier revealed that ALT relative to TERT amplification was associated with longer overall survival in GM patients without metastasis. CONCLUSION: These findings indicate that telomere maintenance mechanisms play a critical role in the tumorigenesis of GMs and may aid in the prediction of clinical prognosis and the development of targeted therapy for the treatment of GM.

Methylation silencing of TGF-ß receptor type II is involved in malignant transformation of esophageal squamous cell carcinoma.

BACKGROUND: Although massive studies have been conducted to investigate the mechanisms of esophageal squamous cell carcinoma (ESCC) carcinogenesis, the understanding of molecular alterations during the malignant transformation of epithelial dysplasia is still lacking, especially regarding epigenetic changes. RESULTS: To better characterize the methylation changes during the malignant transformation of epithelial dysplasia, a whole-genome bisulfite sequencing analysis was performed on a series of tumor, dysplastic, and non-neoplastic epithelial tissue samples from esophageal squamous cell carcinoma (ESCC) patients. Promoter hypermethylation in TGF-ß receptor type II (TGFBR2), an important mediator of TGF-ß signaling, was identified. Further, we evaluated the methylation and expression of TGFBR2 in tumor samples through The Cancer Genome Atlas multiplatform data as well as immunohistochemistry. Moreover, treatment of ESCC cell lines with5-Aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, reactivated the expression of TGFBR2. The lentiviral mediating the overexpression of TGFBR2 inhibited the proliferation of ESCC cell line by inducing cell cycle G2/M arrest. Furthermore, the overexpression of TGFBR2 inhibited the tumor growth obviously in vivo. CONCLUSIONS: The characterization of methylation silencing of TGFBR2 in ESCC will enable us to further explore whether this epigenetic change could be considered as a predictor of malignant transformation in esophageal epithelial dysplasia and whether use of a TGFBR2 agonist may lead to a new therapeutic strategy in patients with ESCC.

Toxic effects of 56Fe ion radiation on the zebrafish (Danio rerio) embryonic development.

All living organisms and ecosystems are permanently exposed to ionizing radiation. Of all the types of ionizing radiation, heavy ions such as 56Fe have the potential to cause the most severe biological effects. We therefore examined the effects and potential mechanisms of iron ion irradiation on the induction of developmental toxicity and apoptosis in zebrafish embryos. Zebrafish embryos at 4h post-fertilization (hpf) were divided into five groups: a control group; and four groups irradiated with 0.5, 1, 2, and 4Gy radiation, respectively. Mortality and teratogenesis were significantly increased, and spontaneous movement, heart rate, and swimming distance were decreased in the irradiated groups, accompanied by increased apoptosis. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, bcl-2, and caspase-3, were significantly affected by radiation exposure. Moreover, protein expression levels of P53 and Bcl-2 changed in accordance with the corresponding mRNA expression levels. In addition, we detected the protein expression levels of γ-H2AX, which is a biomarker for radiation-induced DNA double-strand breaks, and found that γ-H2AX protein levels were significantly increased in the irradiated groups. Overall, the results of this study improve our understanding of the mechanisms of iron ion radiation-induced developmental toxicity and apoptosis, potentially involving the induction of DNA damage and mitochondrial dysfunction. The findings of this study may aid future impact assessment of environmental radioactivity in fish.

Effects of Ru(CO)3Cl-glycinate on the developmental toxicities induced by X-ray and carbon-ion irradiation in zebrafish embryos.

The inhibitory effects of carbon monoxide (CO), generated by Ru(CO)3Cl-glycinate [CO-releasing molecule (CORM-3)], on developmental toxicity in zebrafish embryos induced by ionizing radiation with different linear energy transfer (LET) were studied. Zebrafish embryos at 5h post-fertilization were irradiated with X-ray (low-LET) and carbon-ion (high-LET) with or without pretreatment of CORM-3 1h before irradiation. CORM-3 pre-treatment showed a significant inhibitory effect on X-ray irradiation-induced developmental toxicity, but had little effect on carbon-ion irradiation-induced developmental toxicity. X-ray irradiation-induced significant increase in ROS levels and cell apoptosis could be modified by CORM-3 pretreatment. However, embryos exposed to carbon-ion irradiation showed significantly increase of cell apoptosis without obvious ROS generation, which could not be attenuated by CORM-3 pretreatment. CORM-3 could inhibit apoptosis induced by ionizing radiation with low-LET as an effective ROS scavenger. The expression of pro-apoptotic genes increased significantly after X-ray irradiation, but increased expression was reduced markedly when CORM-3 was applied before irradiation. Moreover, the protein levels of P53 and γ-H2AX increased markedly after X-ray irradiation, which could be modified by the presence of CORM-3. The protective effect of CORM-3 on X-ray irradiation occurred mainly by suppressing ROS generation and DNA damage, and thus inhibiting the activation of P53 and the mitochondrial apoptotic pathway, leading to the attenuation of cell apoptosis and consequently alleviating X-ray irradiation-induced developmental toxicity at lethal and sub-lethal levels.