Synergistic Catalysis of Cobalt Tetroxide and Bamboo-Shaped Carbon Nanotubes Doped with Nitrogen for Oxygen Reduction in Zn-Air Batteries.

Zinc-air batteries (ZABs) have been considered as one of the most emerging systems for energy conversion and storage. However, the preparation of highly efficient oxygen reduction reaction (ORR) catalysts on an air cathode is still faced with significant challenges. Herein, we report a secondary nitrogen source strategy for fine-tuning the active center, which provides a carbon-based hierarchical porous catalyst (termed Co3O4@N/CNT-1000) for highly efficient ORR activity (E1/2 = 0.87 V, JL = 5.32 mA cm-2, and Eonset = 1.021 V) and excellent stability. Controlled experiments demonstrate that such high activity derives from the synergistic effect of cobalt tetroxide and bamboo-shaped carbon nanotubes doped with nitrogen, prepared by the pyrolysis of a two-dimensional metal-organic framework nanosheet (termed NTU-70) and melamine. Furthermore, the ZAB assembled with Co3O4@N/CNT-1000 displays high specific capacity (854 mA h g-1Zn) and power density (179 mW cm-2), excellent long-term cycling (330 h), and durable charging/discharging ability.

The Probiotic Attributes and Anti-pseudorabies Virus Evaluation of Lactobacillus Isolates.

The emergence of pseudorabies virus (PRV) variants brings serious harm to the swine industry, and its effective treatments are limited at present. As one of the probiotics, the Lactobacillus species have beneficial characteristics of regulating the balance of intestinal flora, inhibiting the growth of pathogenic bacteria and viruses' proliferation, and improving self-immunity. In this study, Lactobacillus plantarum HN-11 and Lactobacillus casei HN-12 were selected and identified through morphology observation, Gram stain microscopy, 16S rRNA sequencing analysis, and specific amplification of the recA gene and pheS gene. All tested isolates exhibited rapid adaptation to the different conditions, excellent acid, and bile tolerance, and sensitivity to Salmonella, Staphylococcus aureus, and Escherichia coli. The antibiotic susceptibility assay displayed the isolates sensitive to most antibiotics and resistant to Lincomycin and Norfloxacin. Moreover, the supernatants of HN-11 and HN-12 inhibited PRV proliferation in ST cells. The results of animal experiments showed that supplementing the challenged mice with the supernatants of Lactobacillus isolates in advance delayed the course of the disease. PRV was detected in the heart, liver, spleen, lung, kidney, and brain tissues of dead mice in the test groups, and its copies in the lungs were significantly decreased compared with the control mice (P < 0.05). These findings proved the advantages of L. plantarum and L. casei as potential probiotic cultures, which could provide a basis for its application in microecological preparations and functional formulations.

Detoxification of aflatoxin B1 in broiler chickens by a triple-action feed additive.

The aim of this study was to evaluate the detoxification of aflatoxin B1 (AFB1) in vitro and in broiler chickens using a triple-action compound mycotoxin detoxifier (CMD). Response surface methodology (RSM) was used to evaluate AFB1 detoxification in artificial gastrointestinal fluid (AGIF) in vitro. The AFB1-degradation rate was 41.5% (P < .05) when using a compound probiotic (CP) in which the visible counts of Bacillus subtilis, Lactobacillus casein, Enterococcus faecalis and Candida utilis were 1.0 × 105, 1.0 × 105, 1.0 × 107 and 1.0 × 105 CFU/mL, respectively. When CP was combined with 0.1% AFB1-degrading enzyme to give CPADE, the AFB1-degradation rate was increased to 55.28% (P < .05). The AFB1-removal rate was further increased to above 90% when CPADE was combined with 0.03% montmorillonite to make CMD. In vivo, a total of 150 one-day-old Ross broilers were allotted to 3 groups, 5 replications for each group, 10 broilers in each replication. Group A: basal diet, Group B: basal diet with 40 µg/kg AFB1, Group C: basal diet with 40 µg/kg AFB1 plus CMD. The feeding experiment period was 21 d. The results showed that broiler growth was increased, and AFB1 residues in serum, excreta and liver were decreased by CMD addition in broiler diet (P < .05). In conclusion, CMD was able to remove AFB1 efficiently in vitro and to increase broiler production performance and reduce AFB1 residues in the chickens.

Development of a TB green II-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine circovirus 2 and 3.

Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differentially detect PCV2 and PCV3. The assay specifically detected PCV2 and PCV3, with no fluorescence signals being detected for other non-targeted pig pathogens. The duplex qPCR showed a high degree of linearity (R2 > 0.998), and its limits of detection were 10 and 78 copies/µL for PCV2 and PCV3, respectively. The duplex qPCR could detect and differentiate PCV2 (melting peaks at 85.5 °C) and PCV3 (melting peaks at 82.5 °C), and showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 2.0%. Fifty-six tissue samples from 18 pig farms were used to evaluate the duplex qPCR method. The results revealed infection rates of 66.07% (37/56) and 39.28% (22/56) for PCV2 and PCV3, respectively. The PCV2 + PCV3 co-infection rate was 39.28% (22/56). The developed method could be used as an efficient molecular biology tool for epidemiological investigations of PCV2 and PCV3.

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3.

The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/µL and 61.2 copies/µL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.

Detection and phylogenetic analysis of porcine circovirus type 3 in central China.

Porcine circovirus type 3 (PCV3) is the pathogen responsible for a new infectious disease that was first reported in 2016 in the United States. To further investigate the epidemic profile and genetic diversity of the virus, one hundred and seventy clinical samples (110 tissue samples and 60 serum samples) were collected from 41 different pig farms in 14 cities in central China, and a SYBR Green I-based quantitative real-time PCR method was developed to detect PCV3. The partial cap genes of four field strains from four different farms were sequenced and analysed. The results showed the detection limit was 2.19 × 101 genome copies/µl. Fifty-three of 170 samples were detected as positive for PCV3, giving a PCV3-positive rate of 31.18%, with 48.78% (20/41) of pig farms harbouring PCV3, which varied from 20% to 42.86% between 2013 and 2017. PCV3 could be detected in samples from pigs with different clinical presentations, and the PCV3-positive rates varied for these different clinical presentations. The partial capsid genes of four PCV3 strains (designated YZ, LY-03, NY and SP) shared 96.3%-99.4% nucleotide identity with those available in GenBank. Phylogenetic analysis based on the capsid gene of 32 PCV3 strains showed that the four PCV3 strains in this study were clustered with the China/GD2016 and South Korea Ku-1606 strains. The results of this study will aid our understanding of the molecular epidemiology of PCV3.

Technospheric Mining of Rare Earth Elements from Bauxite Residue (Red Mud): Process Optimization, Kinetic Investigation, and Microwave Pretreatment.

Some rare earth elements (REEs) are classified under critical materials, i.e., essential in use and subject to supply risk, due to their increasing demand, monopolistic supply, and environmentally unsustainable and expensive mining practices. To tackle the REE supply challenge, new initiatives have been started focusing on their extraction from alternative secondary resources. This study puts the emphasis on technospheric mining of REEs from bauxite residue (red mud) produced by the aluminum industry. Characterization results showed the bauxite residue sample contains about 0.03 wt% REEs. Systematic leaching experiments showed that concentrated HNO3 is the most effective lixiviant. However, because of the process complexities, H2SO4 was selected as the lixiviant. To further enhance the leaching efficiency, a novel process based on microwave pretreatment was employed. Results indicated that microwave pretreatment creates cracks and pores in the particles, enabling the lixiviant to diffuse further into the particles, bringing more REEs into solution, yielding of 64.2% and 78.7% for Sc and Nd, respectively, which are higher than the maximum obtained when HNO3 was used. This novel process of "H2SO4 leaching-coupled with-microwave pretreatment" proves to be a promising technique that can help realize the technological potential of REE recovery from secondary resources, particularly bauxite residue.

Ligation of porcine Fc gamma receptor III inhibits levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto macrophages through Fc receptor-mediated endocytosis. FcγR III is the activating receptor with a tyrosine-based activating motif (ITAM) in its cytoplasmic tail, where up-regulates phagocytosis. However, porcine FcγR III's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγR III in PAM cells down-regulated significantly mRNA levels of IFN-α and TNF-α post-pretreatment, and up-regulated significantly mRNA level of IL-10 post-pretreatment, suggesting that porcine FcγR III signal can inhibit the transcriptional levels of innate antiviral cytokine in host cells. Simultaneously, PRRSV infection assay mediated by FcγR III indicated that selective activation of porcine FcγR III in PAM cells inhibited significantly mRNA levels of IFN-α and TNF-α, and enhanced significantly mRNA level of IL-10, and increased significantly viral mRNA levels, in response to PRRSV infection, suggesting that FcγR III ligation can inhibit the antiviral immune response to PRRSV infection. These results elucidated that the one mechanism of PRRSV-ADE regulated via porcine FcγRIII may be by decreasing antiviral cytokine levels, facilitating viral replication.

Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin-6 in mice.

The development of effective vaccines against porcine circovirus type 2 (PCV2) has been accepted as an important strategy in the prophylaxis of post-weaning multisystemic wasting syndrome; a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, DNA vaccines usually induce weak immune responses. In this study, it was found that the efficacy of a DNA vaccine expressing Cap protein was improved by simultaneous expression of porcine IL-6. A plasmid (pIRES-ORF2/IL6) separately expressing both Cap protein and porcine IL-6 was constructed and compared with another plasmid (pIRES-ORF2) expressing Cap protein for its potential to induce PCV2-specific immune responses. Mice were vaccinated i.m. twice at 3 week intervals and the induced humoral and cellular responses evaluated. All animals vaccinated with pIRES-ORF2/IL6 and pIRES-ORF2 developed specific anti-PCV2 antibodies (according to enzyme-linked immunosorbent assay) and a T lymphocyte proliferation response. The percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes were significantly higher in mice immunized with pIRES-ORF2/IL6 than in those that had received pIRES-ORF2. After challenge with the virulent PCV2 Wuzhi isolate, mice vaccinated with pIRES-ORF2/IL6 had significantly less viral replication than those vaccinated with pIRES-ORF2, suggesting that the protective immunity induced by pIRES-ORF2/IL6 is superior to that induced by pIRES-ORF2.

Expression of bioactive porcine interferon-alpha in Lactobacillus casei.

In this study, we constructed an expression cassette containing the inducible lac promoter and the secretion signal from an S-layer protein of Lactobacillus brevis for the expression of porcine interferon-alpha (IFN-α) in Lactobacillus casei (Lb. casei). Reverse-transcriptase PCR verified the presence of porcine IFN-α mRNA in the recombinant Lb. casei. The porcine IFN-α protein expressed in the recombinant Lb. casei was identified by both Western blot analysis and ELISA. We used various pH values and induction times to optimize the yield of IFN-α, and found that induction with 0.8% lactose for 16 h under anaerobic conditions produced the highest concentrations of IFN-α. Furthermore, the activity of porcine IFN-α in the cultural supernatant was evaluated on ST cells infected with pseudorabies virus. The results revealed that porcine IFN-α inhibited virus replication in vitro. The findings of our study indicate that recombinant Lb. casei producing porcine IFN-α has great potential for use as a novel oral antiviral agent in animal healthcare.

Complete genome sequence of a porcine circovirus 2 strain isolated in central china.

We report here the complete genome sequence of the porcine circovirus type 2 (PCV2) XZBK strain, isolated from central China. This is the first report of a member of the 1B subgroup of PCV2b central China. This finding will help in understanding the epidemiology and molecular characteristics of PCV2.

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China.

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003-2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003-2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.

Ligation of porcine Fc gamma receptor I inhibits levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto macrophages through Fc receptor-mediated endocytosis. FcγRI is the activating receptor with a tyrosine-based activating motif (ITAM) in its cytoplasmic tail, where up-regulates phagocytosis. However, porcine FcγRI's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRI in PAM cells down-regulated significantly mRNA levels of IFN-α and TNF-α post-pretreatment, suggesting that porcine FcγRI signal can inhibit the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRI indicated that selective activation of porcine FcγRI in PAM cells inhibited significantly mRNA levels of antiviral cytokine (IFN-α and TNF-α) in response to PRRSV infection, suggesting that FcγRI ligation can inhibit the antiviral immune response to PRRSV infection.

Ligation of Fc gamma receptor IIB enhances levels of antiviral cytokine in response to PRRSV infection in vitro.

PRRSV infection ADE facilitates the attachment and internalization of the virus onto its host cells, such as monocytes and macrophages, through Fc receptor-mediated endocytosis. FcγRIIB is the only inhibitory receptor with a tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, where counters the "ITAM triggered" activation signals and down-regulates phagocytosis. However, porcine FcγRIIB's role in the antiviral immune response to PRRSV infection has not been studied. In this study, our results indicated that selective activation of porcine FcγRIIB in PAM cells up-regulated significantly mRNA levels of IFN-α and TNF-α at any time point post-pretreatment, suggesting that porcine FcγRIIB signal can enhance the innate antiviral response of host cells. PRRSV infection assay mediated by FcγRIIB indicated that selective activation of porcine FcγRIIB in PAM cells enhanced mRNA levels of antiviral cytokine (IFN-α and TNF-α) and repressed mRNA levels of IL-10 in response to PRRSV infection, suggesting that FcγRIIB ligation can enhance the antiviral immune response to PRRSV infection. In addition, FcγRIIB ligation to infection indicated that PRRSV replication in PAM was not positive correlation with increasing of IFN-α mRNA levels and decreasing of IL-10 mRNA levels, suggesting that there is complex viral replication mechanism in immune cells such as PAM for PRRSV.

Construction and immunogenicity of a recombinant fowlpox vaccine coexpressing S1 glycoprotein of infectious bronchitis virus and chicken IL-18.

Infectious bronchitis virus (IBV) poses a major threat to the chicken industry worldwide. In this study, we developed a recombinant fowlpox virus (rFPV) vaccine expressing the IBV S1 gene and chicken interleukin-18 gene (IL-18), rFPV-S1/IL18. Recombinant plasmid pSY-S1/IL18 was constructed by cloning chicken IL-18 into fowlpox virus transfer plasmid containing S1 gene and transfected into the chicken embryo fibroblasts cell pre-infected with S-FPV-017 to generate rFPV-S1/IL18. Expression of the recombinant proteins was confirmed by RT-PCR and IFA. We also constructed the recombinant fowlpox virus rFPV-S1 without IL-18. One-day-old chickens were vaccinated by wing-web puncture with the two rFPVs, and the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels (P<0.05) elicited by either rFPV-S1 or rFPV-S1/IL18. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-S1/IL18 were significantly higher (P<0.05) than in those immunized with rFPV-S1. All chickens immunized with rFPV-S1/IL18 were completely protected (20/20) after challenge with the virulent IBV HN99 strain 43 days after immunization, while only 15 out of 20 of the chickens immunized with the rFPV-S1 were protected. Our results show that the protective efficacy of the rFPV-S1 vaccine could be enhanced significantly by simultaneous expression of IL-18.

[Transcriptional profiles of intestinal pig epithelial cell jejenum 2 cultures infected by vesicular stomatitis virus].

OBJECTIVE: To better understand the host inflammatory responses, in particular inflammatory cytokines responses of Vesicular Stomatitis virus (VSV) infection, and the host-VSV interaction. METHODS: We used VSV Indian strain to infect the Intestinal Pig Epithelial Cell Jejenum (IPEC-J2) cell. Then we measured and analyzed the viral RNA by using real-time PCR. The transcript level of cytokines (IL-2, 6, 8, 10,12, IFN-alpha, IFN-gamma, TNF-alpha, TGF-beta and TLR3) were detected by real-time PCR. RESULTS: We found that the transcript levels of IL-6, IL-8, IL-12, TNF-alpha and TLR3 were increased obviously whereas TGF-beta showed no significant difference. TPEC-J2 cell did not secrete IL-2, IL-10, IFN-alpha and IFN-gamma. CONCLUSION: The level of inflammatory response was increased when VSV infected TPEC-J2 cell.

Cloning, in vitro expression and bioactivity of duck interleukin-18.

The encoding sequence for duck IL-18 was obtained, using reverse transcription-polymerase chain reaction, from mRNA harvested from Con A-stimulated Gushi (GS) duck splenic mononuclear cells. Recombinant duck IL-18 (rduIL-18) was produced in a prokaryotic expression system. In vitro bioactivity of rduIL-18 was determined in a lymphocyte proliferation assay and in vivo bioactivity of rduIL-18 was assessed by addition to a vaccine. Monoclonal antibody (mAb) and polyclonal antibodies (pAbs) specific for rduIL-18 were generated and subsequently characterized by ELISA, Western blot and neutralizing assays. Sequence analysis of GS duck IL-18 demonstrated an open reading frame (ORF) of 603 base pairs encoding for a 200 amino acid precursor protein. The duck encoding sequence shares 85.3% similarity to the chicken equivalent, at the nucleotide level. A His-duIL-18 fusion protein was recognized in Western blot by mAbs against duck and chicken IL-18 (chIL-18), but not by mAb against human IL-18. Recombinant duIL-18 induced in vitro proliferation of Con A-stimulated duck splenocytes and enhanced the immune response of ducks vaccinated with an inactivated oil emulsion vaccine against avian influenza virus. PAb and mAb 5B2 against rduIL-18 had neutralizing ability, inhibiting the biological activities of both recombinant duIL-18 and endogenous duIL-18. The results indicate that rduIL-18 has the potential to be used as an immunoadjuvant, and the mAb against rduIL-18 further facilitates basic immunobiological studies of the role of IL-18 in the avian immune system.

[Construction of eukaryotic express vector of duck interleukin 18 gene and identification of bioactivity of its expressed protein].

Duck IL-18 gene was amplified from plasmid pGEM-DuIL-18 by PCR. The PCR product digested with Pst I and Xho I was inserted into eukaryotic express vector pcDNA3.1(+) to generate an recombinant expression plasmid pcDNA3.1/DuIL-18 (pDuIL-18), and transformed into Escherichia coli JM109. The recombinant colonies were identified by restriction enzyme digestion, PCR and sequencing. DNA sequence confirmed the correct sequence of the recombinant eukaryotic expression plasmid pDuIL-18 in the reading frame and the ligation part. After the transfection of pDuIL-18 into Cos7 cells, duck IL-18 mRNA was expressed in Cos7 cell. The SDS-PAGE analysis showed that the expressed duck IL-18 protein had molecular weight of 23 000 D. The results of methyl thiazolyl tetrazolium (MTT) assay showed that duck IL-18 protein expressed in Cos7 cell could induce significantly transformation of duck T lymphocytes. Immunoenhancement effect of recombinant expression plasmid pDuIL18 on avian influenza vaccine was observed by proliferation response of the T lymphocytes from spleen. It can obviously enhance the cell-mediated immune response.