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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.

To investigate the effects of curcumin on pain threshold and the expressions of nuclear factor κ B (NF-κ B) and CX3C chemokine receptor 1 (CX3CR1) in spinal cord and dorsal root ganglion (DRG) of the rats with sciatic nerve chronic constrictive injury.One hundred and twenty male Sprague Dawley rats, weighing 220-250 g, were randomly divided into 4 groups. Sham surgery (sham) group: the sciatic nerves of rats were only made apart but not ligated; chronic constrictive injury (CCI) group: the sciatic nerves of rats were only ligated without any drug treatment; curcumin treated injury (Cur) model group: the rats were administrated with curcumin 100 mg/(kg·d) by intraperitoneal injection for 14 days after CCI; solvent control (SC) group: the rats were administrated with the solvent at the same dose for 14 days after CCI. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) of rats were respectively measured on pre-operative day 2 and postoperative day 1, 3, 5, 7, 10 and 14. The lumbar segment L4-5 of the spinal cord and the L4, L5 DRG was removed at post-operative day 3, 7 and 14. The change of nuclear factor κ B (NF-κ B) p65 expression was detected by Western blotting while the expression of CX3CR1 was determined by immunohistochemical staining.Compared with the sham group, the TWL and MWT of rats in the CCI group were significantly decreased on each post-operative day (P0.01), which reached a nadir on the 3rd day after CCI, and the expressions of NF-κ B p65 and CX3CR1 were markedly increased in spinal cord dorsal horn and DRG. In the Cur group, the TWL of rats were significantly increased than those in the CCI group on post-operative day 7, 10 and 14 (P0.05) and MWT increased than those in the CCI group on post-operative day 10 and 14 (P0.05). In addition, the administration of curcumin significantly decreased the positive expressions of NF-κ B p65 and CX3CR1 in spinal cord and DRG (P0.05).Our study suggests that curcumin could ameliorate the CCI-induced neuropathic pain, probably through inhibiting CX3CR1 expression by the activation of NF-κ B p65 in spinal cord and DRG.

A non-sporulating, non-motile, catalase- and oxidase-positive, Gram-negative, rod-shaped bacterial strain, designated BA-3T, was isolated from activated sludge of a wastewater treatment facility. The strain was able to degrade about 95 % of 100 mg 3-phenoxybenzoic acid l−1 within 2 days of incubation. Growth occurred in the presence of 0–2 % (w/v) NaCl [optimum, 0.5 % (w/v) NaCl], at pH 5.5–9.0 (optimum, pH 7.0) and at 10–37 °C (optimum, 28 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain BA-3T was a member of the genus Sphingobium ; it showed highest gene sequence similarity to Sphingobium qiguonii X23T (98.2 %), and similarities of Sphingobium species. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) in the cellular fatty acid profile and the DNA G+C content also supported affiliation of the isolate to the genus Sphingobium . Strain BA-3T showed low DNA–DNA relatedness values (21.3±0.8 %) with Sphingobium qiguonii X23T. Based on phenotypic, genotypic and phylogenetic data, strain BA-3T represents a novel species of the genus Sphingobium , for which the name Sphingobium jiangsuense sp. nov. is proposed; the type strain is BA-3T ( = CCTCC AB 2010217T = KCTC 23196T = KACC 16433T).

A novel facultatively anaerobic, non-spore-forming, non-motile, catalase- and oxidase-positive, Gram-negative and rod-shaped bacterial strain, designated Y12T, was isolated from activated sludge of a wastewater bio-treatment facility. The strain was able to degrade about 90 % of added propanil (100 mg l−1) within 3 days of incubation. Growth occurred in the presence of 0–4.5 % (w/v) NaCl (optimum 0.5 %), at 10–40 °C (optimum 28 °C) and at pH 5.5–10.0 (optimum pH 7.0). Vesicular internal membrane structures and photoheterotrophic growth were not observed. The major respiratory quinone was ubiquinone-10 and the major cellular fatty acid was summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). The genomic DNA G+C content of strain Y12T was 63.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Y12T was a member of the genus Catellibacterium, as it showed highest sequence similarities to Catellibacterium caeni DCA-1T (99.1 %) and Catellibacterium. Strain Y12T showed low DNA–DNA relatedness values with C. caeni DCA-1T. Based on phenotypic, genotypic and phylogenetic properties, strain Y12T represents a novel species of the genus Catellibacterium, for which the name Catellibacterium nanjingense sp. nov. is proposed. The type strain is Y12T ( = CCTCC AB 2010218T = KCTC 23298T). An emended description of the genus Catellibacterium is also presented.

A Gram-negative, non-motile, non-spore-forming, non-flagellated rod capable of degrading cypermethrin, designated LQY-18T, was isolated from activated sludge of a wastewater treatment plant in China. Strain LQY-18T grew at 8–40 °C (optimum 30 °C), at pH 5.0–10.0 (optimum pH 7.0) and with 0–5 % (w/v) NaCl (optimum 1 %). The predominant menaquinone was MK-7 (97 %) and the major fatty acids were summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c), iso-C15 : 0 and iso-C17 : 0 3-OH. The DNA G+C content was 40.3 mol%. Phylogenetic analysis revealed that the isolate belonged to the genus Sphingobacterium of the phylum Bacteroidetes and showed low 16S rRNA gene sequence similarity with recognized members of the genus Sphingobacterium. The closest neighbour was Sphingobacterium mizutaii ATCC 33299T (92.9 % 16S rRNA gene sequence similarity). On the basis of phenotypic, genetic and phylogenetic data, strain LQY-18T ( = ACCC 05410T = CCTCC AB 2010005T = KCTC 23009T) should be classified as a representative of a novel species of the genus Sphingobacterium, for which the name Sphingobacterium wenxiniae sp. nov. is proposed.

A synthetic pyrethroid (SP)-degrading bacterium, designated pyd-1, was isolated from SPcontaminated soil. Based on its phenotypic and genotypic properties, the strain was identified as Ochrobactrum tritici. Strain pyd-1 was able to degrade a wide range of SPs, and its degradation efficiencies were dependent on the molecular structure of the SP. Interestingly, the strain degraded cis- and trans-permethrin (cypermethrin) at nearly the same rate and possessed approximately equal hydrolysis activities toward the two enantiomers of fenpropathrin. These results suggest that different isomers of SPs are degraded with equal efficiency by strain pyd-1. We studied the metabolic pathway of fenpropathrin degradation in strain pyd-1 by metabolite identification and enzymatic analysis. Fenpropathrin is degraded by hydrolysis of the carboxylester linkage to yield 2,2,3,3-tetramethylcyclopropanecarboxylic acid and 3-phenoxybenzaldehyde, which is converted to 3-phenoxybenzoic acid (PBA). PBA is further metabolized to 4-hydroxy-3-phenoxybenzoic acid (4-hydroxy-PBA). 4-Hydroxy-PBA is oxidized to protocatechuate and p-hydroquinone. Protocatechuate is further oxidized through an ortho-cleavage pathway, and p-hydroquinone is degraded via 1,2,4-benzenetriol.

A bacterial strain, pcnb-21, capable of degrading pentachloronitrobenzene (PCNB) under aerobic and anoxic conditions, was isolated from a long-term PCNB-polluted soil by an enrichment culture technique and identified as Labrys portucalensis based upon its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis. Effects of different factors, such as temperature and pH, on PCNB biodegradation were studied. Strain pcnb-21 efficiently degraded PCNB at temperatures from 20 to 30 °C and initial pH values from 4 to 7, which might be the first time that a Labrys strain was found capable of efficiently degrading PCNB. The degradation of PCNB was affected by oxygen, and the degradation decreased with increasing aeration. Exogenous electron donors such as glucose, lactic acid and succinic acid promoted the biodegradation of PCNB, while electron acceptors such as sodium nitrite, sodium sulfate, sodium nitrate and sodium sulfate inhibited PCNB biodegradation. The degradation of PCNB in sterile and non-sterile soils by a green fluorescent protein (GFP)-labeled strain, pcnb-21- gfp , was also studied. Cells of pcnb-21- gfp efficiently degraded 100 mg kg −1 PCNB in sterile and non-sterile soils and could not be detected after 42 days. Strain pcnb-21 might be useful in bioremediating PCNB-polluted soils and environment.

OBJECTIVE: To investigate the effects of curcumin on the behavior of chronic constrictive injury (CCI) rats and the CX3CR1 expression in spinal cord dorsal horn and dorsal root ganglia (DRG). METHOD: Seventy-two male SD rats were randomly divided into 4 groups: 1) Sham operation group (Sham); 2) Chronic constrictive injury group (CCI); 3) Curcumin treated group (Cur), administrated with curcumin 100 mg x kg(-1) x d(-1) ip for 14 days after CCI; 4) Solvent contrast group (SC), administrated with an equal volume of solvent for 14 days after CCI. Paw thermal withdrawal (PTWL) and paw mechanical withdrawal threshold (PMWT) were measured on 2 pre-operative and 1, 3, 5, 7, 10, 14 post-operative days respectively. The lumbar segments L4-5 of the spinal cord and the L4, L5 DRG were removed at 3, 7, 14 days after surgery. The expression of CX3CR1 was determined by immunohistochemical staining. RESULT: Compared with Sham group, PTWL and PMWT in CCI group were significantly lower on each post-operative day (P

A butachlor-degrading strain, designated FLY-8, was isolated from rice field soil and was identified as Paracoccus sp. Strain FLY-8 could degrade and utilize six chloroacetamide herbicides as carbon sources for growth, and the degradation rates followed the order alachloracetochlorpropisochlorbutachlorpretilachlormetolachlor. The influence of molecular structure of the chloroacetamide herbicides on the microbial degradation rate was first analyzed; the results indicated that the substitutions of alkoxymethyl side chain with alkoxyethyl side chain greatly reduced the degradation efficiencies; the length of amide nitrogen's alkoxymethyl significantly affected the biodegradability of these herbicides: the longer the alkyl was, the slower the degradation efficiencies occurred. The phenyl alkyl substituents have no obvious influence on the degradation efficiency. The pathway of butachlor complete mineralization was elucidated on the basis of the results of metabolite identification and enzyme assays. Butachlor was degraded to alachlor by partial C-dealkylation and then converted to 2-chloro-N-(2,6-dimethylphenyl)acetamide by N-dealkylation, which subsequently transformed to 2,6-diethylaniline, which was further degraded via the metabolites aniline and catechol, and catechol was oxidized through an ortho-cleavage pathway. This study highlights an important potential use of strain FLY-8 for the in situ bioremediation of chloroacetamide herbicides and their metabolite-contaminated environment.

A synthetic pyrethroid (SP)-degrading bacterial strain, designated JZ-1T, was isolated from activated sludge of a SP-manufacturing wastewater treatment facility and studied using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-1T belonged to the genus Sphingobium, showing highest sequence similarities to Sphingobium faniae DSM 21829T (98.6 %), Sphingobium cloacae JCM 10874T (98.5 %), Sphingobium vermicomposti DSM 21299T (97.4 %) and Sphingobium ummariense CCM 7431T (96.9 %). The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C18 : 1ω7c, C19 : 0 cyclo ω8c, 11 methyl C18 : 1ω7c, C16 : 0 and C14 : 0 2-OH, and the G+C content of the genomic DNA also supported the affiliation of the strain with the genus Sphingobium. Strain JZ-1T showed low DNA–DNA relatedness values with S. faniae DSM 21829T (30.2 %), S. cloacae JCM 10874T (23.3 %), S. vermicomposti DSM 21299T (10.9 %) and S. ummariense CCM 7431T (7.9 %). Based on its phylogenetic position and its phenotypic and genotypic properties, strain JZ-1T represents a novel species of the genus Sphingobium, for which the name Sphingobium wenxiniae sp. nov. is proposed. The type strain is JZ-1T ( = CGMCC 1.7748T = DSM 21828T).