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Table S5

Purpose:Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear.Experimental Design:A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study.Results:A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone.Conclusions:Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Potential protection against the neurotoxic damages of high levels of fluoride on rats and SH-SY5Y cells by extract of Ginkgo biloba leaves, as well as underlying mechanisms, were examined.The rats were divided randomly into 4 groups, i.e., control, treatment with the extract (100 mg/kg body weight, gavage once daily), treatment with fluoride (50 ppm FThe rats with prolong treatment of fluoride exhibited dental fluorosis, the increased contents of fluoride in brains and urine and the declined ability of learning and memory. In the hippocampus of the rats and SH-SY5Y cells exposed to fluoride, the levels of ROS, MDA, apoptosis, 8-OHdG and the protein expressions of histone H2A.XGinkgo biloba extract attenuated neurotoxic damages induced by fluoride exposure to rats and SH-SY5Y cells and the underlying mechanism might involve the inhibition of PARP-1 and the promotion of SIRT1.

The Dongyang gold deposit is a newly discovered epithermal deposit in Fujian Province, Southeast China, along the Circum-Pacific metallogenic belt. Herewith, the authors present mineralogical, scanning electron microscope, and laser ablation inductively coupled clasma mass spectrometry analysis to reveal the relations between Au and Te, As, S, Fe, etc., and discuss the gold precipitation process. The pyrites in this deposit are Fe-deficient, and are enriched in Te and As. The authors infer that As was mainly in form of As-complexes, and Te-Au-Ag inclusions/solid solution also exsits in the Py I. Along with the depletion of Te and As, they were less active chemically in the Py II, and Au may be incorporated into As-rich and Fe-deficient surface sites by chemisorption onto As-rich growth surfaces. Because of the incorporation of new fluid, Te and As became the most active chemically in the Py III, which was the main elements precipitation stage, and As dominantly substituted for S in the lattice of pyrite, due to the more reducing condition. The authors propose Au was in form of invisible gold, and the incorporation of gold can be considered as post-pyrite event, while the Au-bearing minerals were result of post incorporation of gold in arsenian pyrite.

Purpose: Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. Experimental Design: A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. Results: A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. Conclusions: Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Background: Patients with liver cirrhosis are at higher risk of developing hepatocellular carcinoma (HCC). Due to the low accuracy of current methods, new non-invasive strategies for early HCC diagnosis in cirrhotic patients are needed. Methods: A case-control study recruited a total of 2,250 patients with liver cirrhosis (LC), 508 with HCC, and 476 healthy controls (CTRL), from 13 hospitals in 11 provinces of China. Four genomic features of cell-free DNA (cfDNA), including nucleosome footprint, fragmentation, base mismatch and 5-hydroxymethylcytosines were profiled. Training and validation cohorts helped develop the integrated diagnostic model, and a test cohort was used to evaluate its performance. Findings: We established a classifier of each feature for HCC against non-HCC (cirrhosis & healthy control). By integrating these four genomic features, we developed a novel diagnostic model (HIFI, 5-Hydroxymethylcytosines/base mIsmatch/Fragmentation/nucleosome footprInt), and achieved a higher accuracy in differentiating HCCs from liver cirrhosis (AUC=0.997 [0.994-0.999], sensitivity 95.42%, specificity 97.67% in test set) than AFP or PIVKA-II, irrespective of the following demographics and clinical features, age, HBV status, Child-Pugh score, BCLC stage, tumor size, and AFP status. In AFP positive (AFP>20ug/L) liver cirrhosis patients and AFP/PIVKA-II negative (AFP 400μg/L: 100% in liver cirrhosis patients; AFP