Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

The response of anaerobic ammonium oxidation process to bisphenol-A: Linking reactor performance to microbial community and functional gene.

As a typical endocrine disruptor, bisphenol A (BPA) has been widely detected in various water bodies. Although the influence of BPA on traditional biological treatment system has been investigated, it is not clear whether it has potential impact on anaerobic ammonium oxidation (anammox) process. The short- and long-term influences of BPA on reactor operational performance, sludge characteristics and microbial community were investigated in this study. Results revealed that 1 and 3 mg L-1 BPA exhibited a limited adverse impact on granular sludge reactor performance. However, exposure of sludge under 10 mg L-1 BPA would cause an obvious inhibition on nitrogen removal rate from 10.3 ± 0.2 to 7.6 ± 0.4 kg N m-3 d-1. BPA would affect granular sludge metabolic substance excretion and lead to effluent dissolved organic content increase. Both the microbial community and redundancy analysis showed that BPA exhibited a negative influence on Ca. Kuenenia but a positive correlation with SBR1031. Low BPA concentration appeared a limited impact on functional genes while 10 mg L-1 BPA would cause decline of hzsA and hdh abundances. The results of this work might be valuable for in-depth understanding the potential influence of endocrine disruptor on anammox sludge.

Molecular spectroscopy and docking simulation revealed the binding mechanism of phenol onto anammox sludge extracellular polymeric substances.

The rapid development of chemical industry has induced to the large amount of phenolic wastewater production. When the promising anaerobic ammonium oxidation (anammox) was employed to treat the industrial wastewater, phenolic compounds would possibly inhibit the microbial performance. Extracellular polymeric substances (EPSs) play an essential role in protecting cells from being intoxicated by phenolic compound while the distinct mechanism remains elusive. In this work, the interaction of phenol with anammox sludge EPSs and transmembrane ammonium transport (Amt) domain was explored at molecular level by using spectral method and molecular docking simulation. It was found that phenol statically quenched the fluorescent components of EPSs and the protein component dominated the interaction between EPSs and phenol. The overall interaction was an entropy-driven process with hydrophobic interaction as the main driving force, and the CO vibration responded preferentially. As phenol continued to penetrate into the cell surface, there were hydrogen bond, hydrophobic interaction force and π-π base-stacking forces between the Amt domain and phenol. The interaction between phenol and amino acid residues of the Amt domain would interfere the NH4+ transport and further affect the activity of anammox sludge. This work is beneficial for in-depth understanding the role of EPSs in protecting anammox sludge from inhibiting by phenolic pollutants.

Molecular Insight into the Binding Property and Mechanism of Sulfamethoxazole to Extracellular Proteins of Anammox Sludge.

Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 104 M-1. The overall binding was an enthalpy-driven process, with ΔH = -75.15 kJ mol-1, ΔS = -0.175 kJ mol-1 K-1, and ΔG = -21.10 kJ mol-1 at 35 °C. The O-P-O and C═O groups responded first under the disturbance of sulfamethoxazole. Excessive sulfamethoxazole (20 mg L-1) would decrease the ratio of α-helix/(ß-sheet + random coil) of extracellular proteins, resulting in a loose structure. Molecular docking and dynamic simulation revealed that extracellular proteins would provide abundant sites to bind with sulfamethoxazole, through hydrogen bond and Pi-Akyl hydrophobic interaction forces. Once sulfamethoxazole penetrates into the cell surface and combines with the transmembrane ammonium transport domain, it may inhibit the NH4+ transport. Our findings enhance the understanding on the interaction of extracellular proteins and sulfamethoxazole, which may be valuable for deciphering the response property of anammox sludge under the antibiotic stress.

Controllable Synthesis of Two Isomers 4H-Chromene and 2,8-Dioxabicyclo[3.3.1]nonane Derivatives under Catalyst-Free Conditions.

A one-pot method for the selective synthesis of two isomers 4H-chromene and 2,8-dioxabicyclo[3.3.1]nonane derivatives was developed without a catalyst and using EtOH/H2O (4:1, v/v) as the solvent. The reaction was conducted under mild conditions, with forming multiple chemical bonds in one pot and high atom economy, and only a stoichiometric amount of H2O is produced as the byproduct. Its selectivity was controlled by thermodynamics and kinetics, and the reasons for the transformation of the two structures are also discussed.

[Amplification and expression of human Artemin cDNA].

The cDNA fragment encoding human Artemin was amplified by RT-PCR with human fetal brain RNA as template. Result of sequencing showed that the homology of nucleotides is 99.7% between the amplified human Artemin cDNA and the reported one (GenBank accession No. AF115765) and the homology of amino acids is 100%. The prokaryotic expressing vector pGEX-6p-1-hART carrying the amplified DNA fragment was obtained by ligation with the plasmid pGEX-6p-1. The expression of recombinant human Artemin fusion protein in E.coli was analyzed by SDS-polyacarylamide gel electrophoresis. Result demonstrated that the recombinant protein, mostly found in inclusion bodies, accounted for 18.32% of the total bacterial lysate. The inclusion body was dissolved and renatured by the oxidoreduction system, and the recombinant protein is analyzed by Western blotting. Human Artemin cDNA was amplified and the recombinant protein was expressed in vitro successfully.

[Expression, purification of recombinant Luxi yellow cattle IFN-alpha fusion protein and its antiviral activities].

Interferon a gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a( + ) to make a recombinant plasmid pET32a( + )/BoIFN-alpha. The expression of BoIFN-alpha in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-alpha gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-alpha subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40 kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5 x 10(5) u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-alpha C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.

Identification of chicken anemia virus putative intergenotype recombinants.

Chicken anemia virus (CAV), the only member of the genus Gyrovirus in the family Circoviridae is the pathogen of chicken infectious anemia. It is unknown whether homologous recombination happens between CAV strains. In order to gain insight into this matter, we have performed a phylogenetic analysis of full-length CAV strains isolated to detect possible recombination events. Two putative recombinants, SD24, SD22 and the putative parental-like strains were identified with the use of SimPlot program. The two mosaic CAV consist of a novel genotype in the phylogenetic tree. It suggests that homologous recombinant plays roles in generating genetic diversity in natural populations of CAV.

[Relationship between the melanocortin receptor 1 (MC1R) gene and the coat color phenotype in cattle].

The MC1R gene is related to coat color in cattle and to lactoprotein content in milk. Polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing were used to analyze the MC1R of China Holstein Black-white Cattle, China Holstein Red-white Cattle, Luxi Yellow Cattle and Bohai Black Cattle. Three alleles (ED, E+ and e) were identified. Most China Holstein Black-white Cattle had ED and E+ alleles (ED = 0.12, E+ = 0.80). The same was true for Bohai Black Cattle (ED = 0.52, E+ = 0.47). On the other hand, most China Holstein Red-white Cattle and Luxi Yellow Cattle had the e allele (e/e = 0.95). E+/e genotype was present in China Holstein Red-white Cattle and Luxi Yellow Cattle. We reasoned that ED and E+ lad to the synthesis of the black pigment. Furthermore, we found an important SNP (Single Nucleotide Polymorphism) at 725 position of the MC1R coding region.

Preparation and evaluation of anti-SARS coronavirus IgY from yolks of immunized SPF chickens.

Severe acute respiratory syndrome (SARS) is a recently discovered viral disease, characterized by fever, cough, acute fibrinous pneumonia and high infectivity. Specific pathogen-free (SPF) chickens were immunized with inactivated SARS coronavirus and their eggs were harvested at regular intervals. Yolk immunoglobulin (IgY) was extracted using the water dilution method, followed by further purification on a Sephadex G-75 column. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and neutralization test results showed that the IgY obtained was of a high purity and had a strong reactive activity with a neutralization titer of 1:640. Lyophilization and stability tests showed that lyophilized anti-SARS coronavirus IgY had promising physical properties, with no significant reduction in reactive activity and good thermal stability. All these data suggest that the anti-SARS coronavirus IgY could be a new useful biological product for specific antiviral therapy against SARS.

[Microsatellite marker linked with stripe rust resistant gene Yr9 in wheat].

SSR analysis was performed using a wheat near-isogenic line (NIL) Taichuang29 * 6/ Lovrin13, which carried the resistance gene Yr9 against wheat stripe rust and its recurrent parent Taichung29 as materials. After screening with 32 SSR primers on 1B chromosome, reproducible polymorphic DNA fragment amplified by Xgwm582 was identified. Genetic linkage was tested in 177 segregating F2 plants. The results indicated that microsatellite marker Xgwm582 was linked with gene Yr9 resistant to wheat stripe rust. A genetic distance of 3. 7 cM was calculated.

[Construction and functional analysis of the pepB gene disruptant in Aspergillus niger].

An integration plasmid pMW1-pepB for the pepB gene disruption in Aspergillus was constructed. The plasmid contained the pepB gene upstream (P) 1.4kb and downstream (T) 1.3kb homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. P and T DNA fragments were synthesized by PCR from Aspergillus niger chromosomal DNA. The integration plasmid was digested with the Hpa I restriction enzyme, the resultant 4.2kb linear fragment was introduced into the Aspergillus niger strain GICC2773 which expressing the glucoamylase/laccase fusion protein by PEG-mediated transformation. 62 Hygromycin resistance transformants were screened, and from them one strain named pepB29 was identified to be the pepB disruptant by PCR analysis. Data of functional assay of the pepB29 strain indicated that the disruption of the pepB gene secreted reduced acid proteolytic activity, and improved the heterologous protein laccase production.