[High-level expression of anti FLAG tag antibody in plants].

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.

Ethyl Gallate Inhibits Bovine Viral Diarrhea Virus by Promoting IFITM3 Expression, Lysosomal Acidification and Protease Activity.

Bovine viral diarrhea virus (BVDV) is a highly contagious viral disease which causes economic losses to the cattle industry. Ethyl gallate (EG) is a phenolic acid derivative which has various potentials to modulate the host response to pathogens, such as via antioxidant activity, antibacterial activity, inhibition of the production of cell adhesion factors, and so on. This study aimed to evaluate if EG influences BVDV infection in Madin-Darby Bovine Kidney (MDBK) cells, and to understand the antiviral mechanism. Data indicated that EG effectively inhibited BVDV infection by co-treatment and post-treatment in MDBK cells with noncytotoxic doses. In addition, EG suppressed BVDV infection at an early stage of the viral life cycle by blocking entry and replication steps but not viral attachment and release. Moreover, EG strongly inhibited BVDV infection by promoting interferon-induced transmembrane protein 3 (IFITM3) expression, which localized to the cytoplasm. The protein level of cathepsin B was significantly reduced by BVDV infection, whereas with treatment with EG, it was significantly enhanced. The fluorescence intensities of acridine orange (AO) staining were significantly decreased in BVDV-infected cells but increased in EG-treated cells. Finally, Western blot and immunofluorescence analyses demonstrated that EG treatment significantly enhanced the protein levels of autophagy markers LC3 and p62. Chloroquine (CQ) significantly increased IFITM3 expression, and Rapamycin significantly decreased it. Thus, EG may regulate IFITM3 expression through autophagy. Our results showed that EG could have a solid antiviral activity on BVDV replication in MDBK cells via increased IFITM3 expression, lysosomal acidification, protease activity, and regulated autophagy. EG might have value for further development as an antiviral agent.

Microbial community composition and cooccurrence patterns driven by co-contamination of arsenic and antimony in antimony-mining area.

In the current study, a typical Sb mine was selected to explore the microbial community composition and assembly driven by the cocontamination of As/Sb with geographic distance. Our results showed that environmental parameters, especially pH, TOC, nitrate, total and bioavailable As/Sb contents largely affected the microbial community diversity and composition. The total and bioavailable As/Sb levels were significantly positively correlated with the relative abundance of Zavarzinella, Thermosporothrix and Holophaga, while the pH presented a significant negative correlation with the three genera, potentially implying they are important taxonomic groups in acid-mining soils. The cooccurrence network analysis indicated the environmental stress dominated by pH and As/Sb co-contamination affected the microbial modularity and interaction. Meanwhile, Homogeneous selection (HoS, 26.4-49.3%), and drift and others (DR, 27.1∼40.2%) were the most important assembly processes for soil bacterial, and the importance of HoS decreased and the DR increased with geographic distance to the contamination source respectively. Soil pH, nutrient availability, total and bioavailable As/Sb contents significantly affected the HoS and DR processes. This study provides theoretical support for microbial remediation in metal(loid)-contaminated soils.

Arsenic Transformation in Soil-Rice System Affected by Iron-Oxidizing Strain (Ochrobactrum sp.) and Related Soil Metabolomics Analysis.

Iron-oxidizing bacteria (FeOB) could oxidize Fe(II) and mediate biomineralization, which provides the possibility for its potential application in arsenic (As) remediation. In the present study, a strain named Ochrobactrum EEELCW01 isolated previously, was inoculated into paddy soils to investigate the effect of FeOB inoculation on the As migration and transformation in paddy soils. The results showed that inoculation of Ochrobactrum sp. increased the proportion of As in iron-aluminum oxide binding fraction, which reduced the As bioavailability in paddy soils and effectively reduced the As accumulation in rice tissues. Moreover, the inoculation of iron oxidizing bacteria increased the abundance of KD4-96, Pedosphaeraceae and other bacteria in the soils, which could reduce the As toxicity in the soil through biotransformation. The abundance of metabolites such as carnosine, MG (0:0/14:0/0:0) and pantetheine 4'-phosphate increased in rhizosphere soils inoculated with FeOB, which indicated that the defense ability of soil-microorganism-plant system against peroxidation caused by As was enhanced. This study proved that FeOB have the potential application in remediation of As pollution in paddy soil, FeOB promotes the formation of iron oxide in paddy soil, and then adsorbed and coprecipitated with arsenic. On the other hand, the inoculation of Ochrobactrum sp. change soil microbial community structure and soil metabolism, increase the abundance of FeOB in soil, promote the biotransformation process of As in soil, and enhance the resistance of soil to peroxide pollution (As pollution).

The proteasomal inhibitor MG132 increases the efficiency of mouse embryo production after cloning by electrofusion.

In this study, we cloned mice from ES cells by a post-electrofusion MG132 treatment and improved development of cloned embryos with a sequential cultivation protocol. When 5 microM MG132, a proteasome inhibitor, were used to treat the reconstructed embryos, the capacity of in vitro development, implantation and full-term development were significantly improved. Blastocyst formation rates of the reconstructed embryos from X4 ES cells (F1 strain derived from C57BL/6 x 129sv) and J1 ES cells obtained with or without MG132 treatment were 66.9% and 26.6%, and 66.1% and 34.5% respectively (P < 0.05). A total of 146 two-cell embryos cloned from X4 ES cells with MG132 treatment were transferred to recipients, and five cloned pups (3.4%) were born, of which four survived. When the same numbers of two-cell embryos cloned from X4 ES cells without MG132 treatment were transferred, however, no live-born mice were obtained. When embryos cloned from J1 ES cells without MG132 treatment were cultured in KSOM medium for 54 h followed by culture in CZB medium containing 5.6 mM glucose for 42 h, the blastocyst rate was significantly higher than when they were cultured in KSOM continuously for 96 h (34.5% vs 17.1%). However, sequential cultivation did not improve the development of embryos cloned with MG132 treatment and that of parthenotes. In conclusion, MG132 treatment increased the developmental potential of reconstructed mouse embryos, and sequential cultivation improved development of the embryos cloned by electrofusion without MG132 treatment.

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos.

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.