Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines.

BACKGROUND: Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM: To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS: We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS: The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION: Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.

Heavy metal accumulation in soils, plants, and hair samples: an assessment of heavy metal exposure risks from the consumption of vegetables grown on soils previously irrigated with wastewater.

It is common knowledge that soils irrigated with wastewater accumulate heavy metals more than those irrigated with cleaner water sources. However, little is known on metal concentrations in soils and cultivars after the cessation of wastewater use. This study assessed the accumulation and health risk of heavy metals 3 years post-wastewater irrigation in soils, vegetables, and farmers' hair. Soils, vegetables, and hair samples were collected from villages previously irrigating with wastewater (experimental villages) and villages with no history of wastewater irrigation (control villages). Soil samples were digested in a mixture of HCL/HNO3/HCLO4/HF. Plants and hair samples were digested in HNO3/HCLO4 mixture. Inductive coupled plasma-optical emission spectrometer (ICP-OES) was used to determine metal concentrations of digested extracts. Study results indicate a persistence of heavy metal concentration in soils and plants from farms previously irrigated with wastewater. In addition, soils previously irrigated with wastewater were severely contaminated with cadmium. Hair metal concentrations of farmers previously irrigating with wastewater were significantly higher (P < 0.05) than farmers irrigating with clean water, but metal concentrations in hair samples of farmers previously irrigating with wastewater were not associated with current soil metal concentrations. The study concludes that there is a persistence of heavy metals in soils and plants previously irrigated with wastewater, but high metal concentrations in hair samples of farmers cannot be associated with current soil metal concentrations.

[Study on terahertz spectra of multi-walled carbon nanotubes].

In the present paper, the authors report the characterization of multiwalled carbon nanotube at terahertz (THz) frequency range using terahertz time-domain spectroscopy. The surface appearances and microanalysis of multiwalled carbon nanotubes were measured by scanning electron microscope in order to fully understand the unique features and applications of multiwalled carbon nanotube. The results show that the refractive indexes of the sample decrease with increasing frequency in the frequency range of 0.2 to 2 THz, while the absorption coefficients of the sample increase with increasing frequency. In addition, the curve of terahertz absorption coefficients can be fitted by a straight line with a slope of 1.92. From the results of scanning electron microscope, the ranges of inner diameter and outer diameters of the sample were from 5 to 15 nm and from 15 to 25 nm, respectively, and its length was in the order of micrometer. The results of microanalysis of its elemental composition showed that the content of element C was about 94% and the rest were O and Cl elements, which were impurity elements. Mathematical modes of terahertz absorption and refractive indexes in the frequency range of 0.2 to 2.0 THz were established after taking into account Taylor expansion and Maxwell's equations, and the calculation was in relatively good agreement with the observed values of the sample in general. The terahertz refractive indexes and absorption properties of multiwalled carbon nanotube were mainly attributed to the chemical compositions and molecular weight, and carbon nanotubes with different content of carbon could show different terahertz absorption spectra and disclose different unique functions.