Abstract:Anaerobic ammonium oxidation (Anammox) process is a high-rate nitrogen removal technology that has been applied in sludge dewatering effluents treatment with nitrogen removal rate as high as 9.5 kg/(m³·d). However, due to the slow growth rate of the autotrophic Anammox bacteria and the susceptivity to environmental conditions, the start-up of Anammox process is very long; the operation is unstable; and the nitrogen removal from organic-containing and/or toxicant-containing ammonium-rich wastewaters using Anammox process becomes difficult. Thus, the application of this high-rate process is significantly limited. In this paper, a newly-developed Anammox process with sequential biocatalyst (Anammox biomass) addition was established based on the procedure in fermentation engineering. We introduced the Anammox process with sequential biocatalyst addition on start-up, stable operation and the treatment of organic-containing and toxicant-containing ammonium-rich wastewaters. Results show that supplementing high-activity Anammox biomass into reactors will increase the amount of as well as the ratio of Anammox bacteria. Thus, the innovative Anammox process with sequential biocatalyst addition not only accelerates the start-up course, but also enhances the stability of Anammox process. Furthermore, it overcomes the drawbacks of wastewaters containing high organic content and toxic substances. Therefore, the application of Anammox process may be further enlarged.

Abstract:This paper reviews the effects of physical environments (including light, electric field, ultrasound, magnetic field, microgravity, temperature, mechanical vibration, and heterogeneous nucleation interface) on protein crystal nucleation. The research results are summarized and the possible mechanisms of the effects are discussed. In the end of this review, the application prospects of these physical environments (including coupled environments) in protein crystallization are presented.

Abstract:Estrogen-related receptor α (ERRα) is a key regulator for energy metabolism and adipogenesis. However, its role in lipolysis is unknown. To study the function of ERRα in lipolysis, primary cultured differentiated porcine adipocytes were treated by a specific inverse agonist XCT790 or infected with adenoviral vector expressed ERRα for 48 h, in the absence and/or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the triglyceride (TG) content and the glycerol release into the culture media to analysis the effect of ERRα on lipolysis; Further, we analyzed the expression of PPARγ, perilipin A, p-perilipin A, HSL and ATGL with Western blotting. Here, we found that ERRα significantly increased adipocytes differentiation, TG accumulation and glycerol release. Separately or simultaneously block the PKA and ERK pathway do not significantly altered the effect of ERRα on glycerol release. ERRα significantly up-regulated the proteins expression of PPARγ, perilipin A, HSL and ATGL, while the p-perilipin A protein level was not significantly changed. These findings imply that ERRα could increase lipolysis via up-regulating HSL and ATGL, thereby to supply more FFA as substrate for a larger turnover of cellular triglyceride pool during adipocytes differentiation.

Abstract:Wheat peroxidases 1 (WP1) is the major cationic peroxidase of wheat (Triticum aestivum) grain, which is involved in the development of seeds and an important factor to affect the final processing quality of flour. We constructed a prokaryotic expression vector pET28a-WP1, and transformed it into E. coli host strain T7 Expression. His-tag fused WP1 existed as inclusion body, and the recombinant protein was purified by Ni-NTA resin affinity chromatography under denatured condition. The purity of target protein reached 98%. The recombinant WP1 was refolded by gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-WP1 antiserum was higher than 1:625 000. The result of Western blotting demonstrated that the prepared WP1 polyclonal antibody could be used to detect WP1 with high specificity.

Abstract:When Eshcerichia coli CICIM B0013-030 (B0013, ack-pta, pps, pflB) was used for D-lactate production, succinate and acetate were the main byproducts (as much as 11.9 and 7.1% the amount of lactate respectively). In order to decrease the byproduct levels, we inactivated succinate and acetate synthesis in B0013-030. Two recombinant plasmids containing mutation cassettes of frdA::difGm and tdcDE::difGm respectively were constructed first. The mutation cassettes were used to delete the target genes on the chromosomal by Red recombination. Subsequently, the antibiotic resistance gene was excised from the chromosomal by Xer recombination. Thereby, mutants B0013-040B (B0013-030, frdA) and B0013-050B (B0013-040B, tdcDE) were produced. D-lactate producing abilities of the engineered strains were tested both in shake flasks and in bioreactors using two-phase fermentation (aerobic growth and anaerobic fermentation) with glucose as the sole carbon source. When fermentation was carried out in shake flasks, inactivation of frdA in B0013-030 to produce B0013-040B reduced succinate accumulation by 80.8%. When tested in a 7-liter bioreactor, B0013-040B accumulated 114.5 g/L D-lactate of over 99.9% optical purity. However, 1.0 g/L succinate and 5.4 g/L acetate still remained in the broth. Further inactivation of tdcD and tdcE genes in B0013-040B to produce B0013-050B decreased acetate and succinate accumulation to 0.4 g/L and 0.4 g/L respectively, and lactate titer was as much as 111.9 g/L (tested in the 7-liter bioreactor). In light of the lower byproduct levels and high lactate production, strain B0013-050B may prove useful for D-lactate production.

Abstract:Soil archaea and fungi play important roles in the greenhouse soil ecosystem. To develop and apply rich microbial resources in greenhouse ecological environment, and to understand the interaction between microbes and plants, we constructed archaeal 16S rRNA and fungal 18S rRNA gene libraries to analyze the compositions of archaeal and fungal communityies. Total greenhouse soil DNA was directly extracted and purified by skiving-thawing-lysozyme-proteinase K-SDS hot treatment and treatment of cetyltriethylammnonium bromide (CTAB). After PCR amplification, retrieving, ligating, transforming, screening of white clones, archaeal 16S rRNA and fungal 18S rRNA gene libraries were constructed. The sequences of archaea and fungi were defined into operational taxonomic units (OTUs) when 97% similarity threshold for OTU assignment was performed by using the software DOTUR. Phylogenetic analysis showed that crenarchaeota and unidentified-archaea were the two major sub-groups and only a few of euryarchaeota existed in the archaeal clone library, total 45 OTUs. All the crenarchaeota belonged to thermoprotei; except for Basidiomycotina, the other four sub-group fungi were discovered in the fungal library, total 24 OTUs. The diversities of archaea were very abundant and a few euryarchaeota (methanebacteria) existed in the archaeal clone library, it might be directly related to the long-term high temperature, high humidity, and high content of organic matter. The limitation of oxygen was the other reason for causing this phenomenon; Ascomycotina (over 80%) was the dominant sub-groups in fungal library. It was because most of the plant fungal diseases belonged to soil-borne diseases which gone through the winter by the ways of scierotium or perithecium and became the sources of primary infection.

Abstract:The flocculating yeast strain SPSC01 is a fusant strain of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The use of SPSC01 to absorb Cr(VI) from Cr(VI) containing aqueous solution would greatly reduce the cost of post-adsorption separation, since the superior flocculating property of SPSC01 would allow easy separation of the Cr(VI)-biomass from the solution. In order to investigate the effects of flocculating proteins on Cr(VI) reduction and absorption by SPSC01, the absorption behaviors of SPSC01 and its parental strains were compared. The results showed that Cr(VI) removal rate of SPSC01 was almost the same as that of S. pombe, which also has flocculating ability, but was faster than that of S. cerevisiae, which has no flocculating ability. When the system reached equilibrium, the amount of total Cr adsorbed by S. pombe, SPSC01 and S. cerevisiae were 68.8%, 48.6% and 37.5%, respectively. This showed that flocculation was beneficial to Cr(VI) reduction and adsorption, and suggested that focculating proteins may play a role in enhancing the Cr(VI) adsorption capacity of SPSC01 and S. pombe. We investigated the mechanism of Cr(VI) adsorption by SPSC01 using chemical modification and FTIR. The results indicated that the major functional groups (amino, carboxyl and amide) of surface proteins may contribute to the absorption of Cr(VI).

Abstract:The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5′-untranslated region (UTR) and a 159 bp 3′-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys¹⁸³, His³²² and Asn³⁴³) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases.

Abstract:We investigated the plant regeneration and production of flavonoids in three high-yield flavonods transgenic Saussurea involucrata hairy roots C17, C27 and C46 by quantification of two phytohormones GA₃ and IAA. The results showed that GA₃ concentration at more than 1.0 mg/L could induce adventitious shoots in the hairy root lines. The highest shoot regeneration rate, about 82%, was obtained when the hairy roots C17 were cultured with 2.0 mg/L GA₃. The results on HPLC and UV spectrophotometry showed that exogenous application of both GA₃ and IAA increased the content of flavonoids in the hairy roots. The contents of flavonoids and apigenin in the hormone-treated hariy roots and regenerates were higher comparing with those in the untreated hairy roots and the regenerates. However, the content of flavonoids was not related to tissue weight, and was negatively related to the regeneration efficiency.

Abstract:Plant dehydroascorbate reductase (DHAR) is a physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction. In this study, two DHARs genes (SmDHAR1 and SmDHAR2) were isolated from Selaginella moellendorffii. The SmDHAR1 and SmDHAR2 genes encode two proteins of 218 and 241 amino acid residues, with a calculated molecular mass of 23.97 kDa and 27.33 kDa, respectively. The genomic sequence analysis showed SmDHAR1 and SmDHAR2 contained five and six introns, respectively. Reverse transcription PCR revealed that the SmDHAR1 and SmDHAR2 were constitutive expression genes in S. moellendorffii. The recombinant SmDHAR1 and SmDHAR2 proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The recombinant SmDHAR1 showed 116-fold higher enzymatic activity towards the substrate dehydroascorbate than recombinant SmDHAR2. The recombinant SmDHAR1 showed higher thermal stability than recombinant SmDHAR2. These results indicated obvious functional divergence between the duplicate genes SmDHAR1 and SmDHAR2.

Abstract:To study the chemosensitivity and the mechanisms of recombinant adenovirus vector expressing ING4 and IL-24 (Ad-ING4-IL-24) on lung adenocarcinoma in vitro and in vivo, the expression of ING4 and IL-24 in A549 cells was detected by RT-PCR and Western blotting. The growth inhibition, apoptosis rate and apoptosis body were measured by MTT, flow cytometry and Hoechst staining respectively. For in vivo study, we first established the A549 tumor model by grafting A549 cells in athymic nude mice; and then injected Ad-ING4-IL-24 into the tumors. Two weeks after injection, we killed the mice, removed the tumors, weighted and calculated the ratios of tumor-suppression. We also detected the expressions of ING4, IL-24, bax, bcl-2, VEGF with immunohistochemistry. The results indicated that ING4 and IL-24 were proved successfully transcription and expression in A549 cells. More interestingly, the joint group inhibited the growth of A549 cells and induced apoptosis. The in vivo data showed that the joint group suppressed the tumor growth conspicuously through up-regulating the expression of bax, and down- regulating the expression of bcl-2, VEGF. The study proved that Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung adenocarcinoma, which may related with cell apoptosis and antiangiogenesis.

Abstract:To develop novel and effective HBV therapeutic vaccine, we constructed an expression vector, pVRC-HBSS1, in which PreS1 (21?47aa) coding gene fused to the C-terminal of the S (1?223 aa) coding gene of HBV, and prepared the protein particle vaccine HBSS1 that consist of S and PreS1 fusion antigen derived from CHO system. We immunized mice by priming three times with DNA vaccine via different methods (i.e., intramuscular injection, intradermal injection with electroporation), then boosting once with protein particle vaccine. We analyzed the immune response among various vaccination groups. The higher level of S or PreS1 specific antibodies was detected in the group via intradermal injection with electroporation, compared with that of direct intramuscular injection. We further found that the specific cellular immune responses (IFN-γ ELISpot analysis) in the group priming with DNA vaccines and boosting with protein subunit vaccine particles, was significantly higher than that of the DNA or protein particle subunit alone. Moreover, combination vaccination priming with intradermal injection DNA via electroporation and boosting with protein particle induced the strongest cellular immune response. These results provide a basis for rational design and application of the novel HBV therapeutic vaccine.

Abstract:Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol^®) and other taxoids. In the cell culture of Taxus chinensis, Taxuyunnanine C (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, we employed Real-time Quantitative PCR (RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes (TASY, TDAT, T5αH, TαH, T10βH and T14βH) were quantified under the process condition of double elicitation by 2,3-dihydroxylpropanyl jasmonate (DHPJA) (100 μmol/L on day 7 and day 12), and sucrose feeding (20 g/L) on day 7. This process treatment led to a high accumulation of Tc at (554.46±21.28) mg/L 8 days after the first elicitation. Then 9 days after the second elicitation, Tc production was as high as (997.72±1.51) mg/L. The early pathway genes TASY and TDAT were significantly up-regulated by 182-fold and 98-fold, respectively for the first DHPJA elicitation and by 208-fold and 131-fold, respectively for the second elicitation. The induction occurred after each elicitation lasted for about 24 h before their abundances decreased. Things are somewhat different in the case of the other four genes T5αH, TαH, T10βH and T14βH. For gene TαH, it was highly up-regulated by 3061-fold for the first DHPJA elicitation and by 1016-fold for the second elicitation. For the other three genes T5αH, T10βH, T14βH, they were up-regulated by 13-fold, 38-fold and 20-fold, respectively for the first DHPJA elicitation and by 7-fold, 16-fold and 6-fold, respectively for the second elicitation. The RQ-PCR results showed that there is tight correlation between gene expression and Tc accumulation. Gene expression was in accordance with Tc yield. Elicitation could improve expression of six genes. While along with culture course, high expression of the genes weakened. Elicitation for the second time would promote high expression of the genes again.

Abstract:Glycosylation is vital for activity, higher structure and function of protein. Glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated, while those from mammalian cells contain N-glycan of hybrid or complex type. We introduced the α-1,2-mannosidase I (MDSI) into yeast cells, which catalyzed an essential proceeding of N-glycan structures from Man₈GlcNAc₂ to Man₅GlcNAc₂. The plasmids contained MDSI genes from Homo sapiens [HMDSI(Δ185)] or Arabidopsis thaliana [ATMDSI(Δ48)], and three ER-signals were used to be transformed a mutant Pichia pastoris GJK01 , respectively. The reporter protein HSA/GM-CSF (human serum albumin and granulocyte-macrophage colony stimulating factor fusion protein) was expressed and its N-glycans were analyzed by DSA-FACE (DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis). The plasmid contained ER-ScMnsI-ATMDSI(Δ48) was expressed in Pichia pastoris, the Man₅GlcNAc₂ N-glycan on secreted glycoprotein HSA/GM-CSF was observed. The research reported here provided basic substrate to obtain the hybrid- and complex-type glycans in mammalian cell.