[Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFbeta].

MNSFbeta (Monoclonal nonspecific suppressor factor beta) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (GenBank accession number: KF77642500) was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFbeta which was transfected into swine umbilical vein endothelial cells (SUVECs) using Lipofectamine 2000. The expression of GFP was detected by fluorescence microscopy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta protein was a stable protein consisting of a ubiquitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.

Quercitrin restrains the growth and invasion of lung adenocarcinoma cells by regulating gap junction protein beta 2.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer (NSCLC) with high lethality, and quercitrin exhibits anticancer characteristics. Here, we attempted to uncover the anticancer activity of quercitrin in LUAD. In this work, quercitrin prohibited the cell viability and clone-formation of LUAD cells in vitro. Meanwhile, quercitrin treatment reduced the aggressive phenotypes in LUAD cells. Further, Gap Junction Protein Beta 2 (GJB2) expression was aberrantly higher in LUAD when compared within control tissue. The higher expression of GJB2 is associated with an inferior overall survival for patients with LUAD. Finally, the reintroduction of GJB2 offset the inhibiting influence of quercitrin in LUAD cells. Altogether, these findings disclosed that quercitrin suppressed the growth and metastatic-related traits of LUAD cells partly via regulating GJB2 expression.

Methane emissions responding to Azolla inoculation combined with midseason aeration and N fertilization in a double-rice cropping system.

Methane (CH4) is an important greenhouse gas (GHG), and paddy fields are major sources of CH4 emissions. This pot experiment was conducted to investigate the integrated effects of Azolla inoculation combined with water management and N fertilization on CH4 emissions in a double-rice cropping system of Southern China. Results indicated that midseason aeration reduced total CH4 emissions by 46.9%, 38.6%, and 42.4%, followed by N fertilization with 32.5%, 17.0%, and 29.5% and Azolla inoculation with 32.5%, 17.0%, and 29.5%, on average, during the early, late, and annual rice growing seasons, respectively. The CH4 flux peaks and total CH4 emissions observed in the late rice growing season were significantly higher than those in the early rice growing season. Additionally, CH4 fluxes correlated negatively to soil redox potential (Eh) and dissolved oxygen (DO) concentration. Azolla inoculation and N fertilization greatly increased the rice grain yields, whereas midseason aeration had distinct effects on grain yields in both rice seasons. The highest annual rice grain yields of approximately 110 g pot-1 were obtained in the Azolla inoculation and N fertilization treatments. In terms of yield-scaled CH4 emission, Azolla inoculation combined with midseason aeration and N fertilization generated the lowest yield-scaled CH4 emissions both in the early and in the late rice growing seasons, as well as during the annual rice cycle. In contrast, the highest yield-scaled CH4 emission was obtained in the treatment employed continuous flooding, without Azolla and no N application. Our results demonstrated that Azolla inoculation, midseason aeration, and N fertilization practices mitigated total CH4 emissions by 18.5-42.4% during the annual rice cycle. We recommend that the combination of Azolla inoculation, midseason aeration, and appropriate N fertilization can achieve lower CH4 emissions and yield-scaled CH4 emissions in the double-rice growing system.

Highly pathogenic porcine reproductive and respiratory syndrome virus GP5 B antigenic region is not a neutralizing antigenic region.

In 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) caused great economic losses emerged in China and continues to be a threat for the pig industry. B antigenic region (AR) ((37)SHL/FQLIYNL(45)) of GP5 was considered to be a major linear neutralizing AR in PRRSV classical strains. However, peptide-purified antibodies against this AR did not neutralize PRRSV in a recent report. Compared with classical PRRSV, one amino acid mutation (L/F(39)→ I(39)) was found in B AR of HP-PRRSV. To study the ability of B AR of HP-PRRSV to induce neutralizing antibody (NA) in vitro and in vivo, rabbit antisera against B AR with and without the mutation and pig hyperimmune sera with high titer of NAs against HP-PRRSV were prepared. Immunofluorescence assays (IFA) showed that the two rabbit antisera both had reactivity to classical PRRSV CH-1a and HP-PRRSV HuN4 with no observable difference in IFA titer. However, antisera did not have neutralizing activity against classical PRRSV CH-1a and HP-PRRSV HuN4. No correlation was observed between the levels of anti-B AR peptide antibodies and NAs in pig hyperimmune sera that were detected by indirect ELISA and virus neutralization, respectively. B AR peptide-specific serum antibodies had no neutralizing activity and, GST-B fusion protein could not inhibit neutralization of NAs in pig hyperimmune sera. Based on these findings, we conclude that B AR of HP-PRRSV is not a neutralizing AR of HP-PRRSV GP5.

Isolation of serotype 2 porcine teschovirus in China: evidence of natural recombination.

Porcine teschovirus (PTV), the pathogen of porcine polioencephalomyelitis, is a member of the family Picornaviridae. In this study, a new PTV strain (designated as JF613) was isolated from pigs in China. It was confirmed by the specific CPE on susceptible cells, RT-PCR and nucleotide sequencing. Analysis of its amino acids sequence of complete polyprotein indicated that the isolate belongs to serotype 2. Genetic recombination is a well-known phenomenon for picornavirus which has been demonstrated in many other members of the family, but it remains so far unclear whether recombination occurs in PTV. To detect possible recombination events, 30 sequences of complete coding regions of PTV strains accessible in GenBank were examined. Putative recombinant sequence was identified with the use of SimPlot program. The result showed that the genomic sequence of our isolate exhibited highest similarities with strains of serotypes 2 and 5, respectively, in two crossover regions, suggesting the recombination event in PTV. Then the mosaic structure of viral genome was confirmed by bootscanning and genetic algorithm for recombination detection (GARD). This represents the first PTV-2 isolate in China. Furthermore, our study provided the first evidence of natural recombination in PTV and indicated that homologous recombination may be a driving force in PTV evolution.

Cloning and identification of porcine programmed death 1.

Programmed death 1 (PD-1) is a member of the immunoglobulin (Ig) superfamily, which is expressed on activated T cells, B cells and monocytes. Many researches have demonstrated that a high level of PD-1 expression is closely related to persistent infection and immune evasion in some human infections. In order to study the relationship between PD-1 expression and persistent infections caused by some porcine viruses, we first cloned the porcine PD-1 from porcine PBMCs based on the blast result in the EST database using the human PD-1 sequence. Sequence analysis showed that the cloned PD-1 molecule shares 63 and 54% amino acid sequence identity with human and murine PD-1, respectively. Its molecular structure is also similar to that of human and murine PD-1, containing an IgV-like domain in the extracellular region and two immune regulatory motifs in its cytoplasmic tail. The in vitro T cell proliferation assay showed that the cloned PD-1 could inhibit porcine T cell proliferation by 71% and secretion of IFN-gamma and IL-2 by 64 and 53%, respectively. These data suggest that porcine PD-1 negatively regulates the porcine immune response in a similar manner to that of its counterpart in the human and mouse immune system.