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The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting and puromycin antibiotic selection. In this protocol, we described the detailed procedure for constructing the HBV-replicating stable cells and systematically evaluating HBV replication and viral protein expression profiles of these cells.

The housefly larvae (Musca domestica) vermireactor is capable of highly efficient waste reduction for swine manure management; however, effectiveness of larvae-vermicompost land utilization and the associated impact on soil environment are poorly understood. This study, which integrated incubation pot experiments and field plot tests on a chrysanthemum (Chrysanthemum morifolium) farm, was designed to investigate soil biochemical and microbial functions in response to inorganic fertilizer (NPK), vermicompost (VC) application, and/or combination of inorganic fertilizer and vermicompost (I + V). Pot experiments indicated that available soil nitrogen (N), phosphorus (P), and potassium (K) were significantly higher (21%-43%, 206%-306%, and 35%-36%, respectively) with VC treatment compared to NPK treatment; meanwhile, soil organic carbon (C), N and P mineralization was increased by 46%-57%, 53%-70%, and 30%-113%, respectively. Vermicompost application increased soil highly labile organic C by 19% to 42%. Vermicompost also enhanced soil enzymatic activities: 37%-68% for dehydrogenase, 22%-107% for urease, and 3.4%-56% for phosphatases, but activities of soil β-1,4-glucosidase and β-1,4-N-acetylglucosaminidase were reduced by 17%-53% and 24%-42%, respectively. Compared to pot experiment, continuous land application of vermicompost overwhelmingly caused soil nutrient accumulation, increased soil C stock and microbial biomass, enhanced soil mineralization, and improved C-, N-, and P-related enzymatic activities. Co-inertia analysis indicated that soil fertility indices positively correlated with most microbiological indices. This work reveals VC as a new biofertilizer for crop production and highlights its merit on soil health improvement. [ABSTRACT FROM AUTHOR]

Summary: Vermicomposting using black soldier fly (BSF) larvae (Hermetia illucens) has gradually become a promising biotechnology for waste management, but knowledge about the larvae gut microbiome is sparse. In this study, 16S rRNA sequencing, SourceTracker, and network analysis were leveraged to decipher the influence of larvae gut microbiome on food waste (FW) biodegradation. The microbial community structure of BSF vermicompost (BC) changed greatly after larvae inoculation, with a peak colonization traceable to gut bacteria of 66.0%. The relative abundance of 11 out of 21 metabolic function groups in BC were significantly higher than that in natural composting (NC), such as carbohydrate‐active enzymes. In addition, 36.5% of the functional genes in BC were significantly higher than those in NC. The changes of metabolic functions and functional genes were significantly correlated with the microbial succession. Moreover, the bacteria that proliferated in vermicompost, including Corynebacterium, Vagococcus, and Providencia, had strong metabolic abilities. Systematic and complex interactions between the BSF gut and BC bacteria occurred over time through invasion, altered the microbial community structure, and thus evolved into a new intermediate niche favourable for FW biodegradation. The study highlights BSF gut microbiome as an engine for FW bioconversion, which is conducive to bioproducts regeneration from wastes. [ABSTRACT FROM AUTHOR]

Gram-staining-negative, oxidase-positive, catalase-positive, non-motile, non-spore-forming, and rod-shaped bacterium, designated BJQ-6, was isolated from activated sludge of a waste-water treatment plant in Jiangsu Province, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BJQ-6 belonged to the genus Rhodanobacter, and shared 98.7% similarity with Rhodanobacter lindaniclasticus LMG 18385 and <97% similarities with other Rhodanobacter species. The major fatty acids were iso-C (17.6%), iso-C (19.3%), and Summed feature 9 (isoC ω9c and/or C 10-methyl) (25.8%). The DNA G+C content of strain BJQ-6 was 64.8 mol%. Based on the phenotypic and phylogenetic considerations, strain BJQ-6 represents a novel species of the genus Rhodanobacter, for which the name Rhodanobacter xiangquanii sp. nov. is proposed. The type strain is BJQ-6 (=CCTCC AB 2010106 =KCTC 23100). [ABSTRACT FROM AUTHOR]

Insertion mutagenesis has become one of the most popular methods for gene functions analysis. Here we report a two-element Ac/Ds transposon system containing enhancer trap and gene trap for gene tagging in rice. The excision of Ds element was examined by PCR amplification. The excision frequency of Ds element varied from 0% to 40% among 20 F2 populations derived from 11 different Ds parents. Southern blot analysis revealed that more than 70% of excised Ds elements reinserted into rice genome and above 70% of the reinserted Ds elements were located at different positions of the chromosome in rice. The result of histochemical GUS analysis indicated that 28% of enhancer trap and 22% of gene trap tagging plants displayed GUS activity in leaves, roots, flowers or seeds. The GUS positive lines will be useful for identifying gene function in rice. [ABSTRACT FROM AUTHOR]

Rice paddy soil-associated microbiota participate in biogeochemical processes that underpin rice yield and soil sustainability, yet continental-scale biogeographic patterns of paddy soil microbiota remain elusive. The soil bacteria of four typical Chinese rice-growing regions were characterized and compared to those of nonpaddy soils. The paddy soil bacteria were significantly less diverse, with unique taxonomic and functional composition, and harbored distinct cooccurrence network topology. Both stochastic and deterministic processes shaped soil bacteria assembly, but paddy samples exhibited a stronger deterministic signature than nonpaddy samples. Compared to other environmental factors, climatic factors such as mean monthly precipitation and mean annual temperature described most of the variance in soil bacterial community structure. Cooccurrence network analysis suggests that the continental biogeographic variance in bacterial community structure was described by the competition between two mutually exclusive bacterial modules in the community. Keystone taxa identified in network models ( Anaerolineales , Ignavibacteriae , and Deltaproteobacteria) were more sensitive to changes in environmental factors, leading us to conclude that environmental factors may influence paddy soil bacterial communities via these keystone taxa. Characterizing the uniqueness of bacterial community patterns in paddy soil (compared to nonpaddy soils) at continental scales is central to improving crop productivity and resilience and to sustaining agricultural soils. IMPORTANCE Rice fields provide food for over half of the world's human population. The ecology of paddy soil microbiomes is shaped by human activities, which can have a profound impact on rice yield, greenhouse gas emissions, and soil health. Investigations of the soil bacteria in four typical Chinese rice-growing regions showed that (i) soil bacterial communities maintain highly modularized species-to-species network structures; (ii) community patterns were shaped by the balance of integrated stochastic and deterministic processes, in which homogenizing selection and dispersal limitation dominate; and (iii) deterministic processes and climatic and edaphic factors influence community patterns mainly by their impact on highly connected nodes (i.e., keystone taxa) in networks. Characterizing the unique ecology of bacterial community patterns in paddy soil at a continental scale may lead to improved crop productivity and resilience, as well as sustaining agricultural soils.