Abstract:High-throughput biological technologies are now widely applied in biology and medicine, allowing scientists to monitor thousands of parameters simultaneously in a specific sample. However, it is still an enormous challenge to mine useful information from high-throughput data. The emergence of network biology provides deeper insights into complex bio-system and reveals the modularity in tissue/cellular networks. Correlation networks are increasingly used in bioinformatics applications. Weighted gene co-expression network analysis (WGCNA) tool can detect clusters of highly correlated genes. Therefore, we systematically reviewed the application of WGCNA in the study of disease diagnosis, pathogenesis and other related fields. First, we introduced principle, workflow, advantages and disadvantages of WGCNA. Second, we presented the application of WGCNA in disease, physiology, drug, evolution and genome annotation. Then, we indicated the application of WGCNA in newly developed high-throughput methods. We hope this review will help to promote the application of WGCNA in biomedicine research.

Abstract:Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

Abstract:To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79?18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

Abstract:Protein tyrosine phosphatase (PTP, EC 3.1.3.48) specifically catalyzes the removal of phosphate groups from phosphorylated tyrosine residues, resulting in protein dephosphorylation, thus regulates life activities such as cell growth, proliferation, differentiation and immunization. Protein tyrosine phosphatase h of Bombyx mori (BmPTP-h) is involved in the replication of nucleopolyhedrovirus (NPV) in Bombyx mori, but the structure and properties of BmPTP-h are little known so far. In this study, the coding sequence of BmPTP-h gene was cloned from the midgut of Bombyx mori, and its genomic structure, mRNA structure, sequence signature, secondary structure and the state in solution were analyzed. Homologous amino acid sequences alignment analysis indicated that BmPTP-h had a high similarity to PTP sequences of numbers of insect NPVs, implying that they may have a common ancestor and similar function. We constructed a prokaryotic expression vector, expressed and obtained the soluble recombinant BmPTP-h in Escherichia coli at 25 ℃, and purified BmPTP-h using Ni-NTA affinity chromatography. Gel filtration analysis showed that BmPTP-h was able to form aggregate and monomer in solution. Circular dichroism spectroscopy analysis showed that the recombinant BmPTP-h contained α-helix structure. Increasing temperature resulted in the unfolding of the α-helix structure of BmPTP-h and the decrease of the α-helix structure content of BmPTP-h. These studies provide a basis to better study the structure and regulation mechanism of BmPTP-h.

Abstract:We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.

Abstract:To study the effect of Astragalus polysaccharide (APS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) cultured in hypoxic environment. The optimal APS concentration, which could promote the proliferation of BMSCs, was screened by methyl thiazolyl tetrazolium method. The concentration was used to intervene in BMSCs-induced by adipogenic differentiation fluid growing in different oxygen concentrations (3%, 6%, 10% and 20%). The formation of lipid droplets in the BMSCs-intervened was observed by oil red O staining under the optical microscope. The mRNA and protein levels of the lipid relating genes peroxisome proliferator activated receptor gamma 2 (PPAR-γ2) and lipoprotein lipase (LPL) were detected by Real-time PCR and Western blotting, respectively. The results showed that, comparing with the control group, 40 μg/mL APS could significantly promote the proliferation of BMSCs under low oxygen concentration. A large amount of lipid droplets existed in BMSCs growing in the adipogenic inducing fluid containing 40 μg/mL APS and the hypoxic environment, and the protein and mRNA levels of PPAR-γ2 and LPL also raised. It was worth noting that the phenomenon was more significant in 10% oxygen concentration, and the difference was statistically significant (P<0.05). 40 μg/mL APS had effect on promoting the proliferation and adipogenic differentiation of BMSCs cultured in hypoxic environment, and the effect was related to the concentration of oxygen of BMSCs-cultured.

Abstract:Steady improvement in mass spectrometers technology has transformed the targeted proteome analysis into a new stage. Parallel reaction monitoring (PRM) technology has evolved from the basic multiple reaction monitoring (MRM) targeted proteomics methods in recent years. PRM performs with a higher sensitivity, throughput and reproducibility in targeted quantification, however its limitations in effectiveness and accurate quantification of samples with higher complexity still remain unsolved. In this study through improving the chromatographic conditions of PRM we established a simple and robust platform for targeted proteomic quantification. The newly established PRM system is equipped with columns with increased inner diameter (150 μm) and decreased total length (8 cm); faster liquid phase elution rate (800 nL/min) and shortened elution gradient (35 min). These modifications enable PRM platform to combine with dual reverse phase chromatography, to quantify up to 400 low abundance peptides in human 293T cells whole cell extract. Our findings would benefit the promotion of PRM technology, especially providing a technical option for accurate quantification of low abundance proteins.

Abstract:Isotope Dilution Mass Spectrometry (IDMS) is the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key is getting the corresponding fully uniformly(U) 13C-labeled metabolites to be measured. The conventional procedure for getting fully U 13C-labeled metabolites is through batch cultivation, but intracellular metabolites concentrations by this method are generally low. By applying U 13C-labeled glucose pulse, combined with fast sampling and quenching, mixture of fully U 13C-labeled intracellular metabolites was successfully extracted with higher concentration from Pichia pastoris G/DSEL fed with fully U 13C-labeled glucose as only carbon source. Quantitative results from liquid chromatography tandem mass spectrometry (LC-MS) and gas chromatography tandem mass spectrometry (GC-MS) show that concentrations of organic acids, sugar phosphates, amino acids and nucleotides were 2–10 folds higher than those without glucose pulse. Therefore, the glucose pulse method can efficiently improve the usage of fully U 13C-labeled glucose converting to 13C-labeled metabolites, and achieve the detection of intracellular metabolites with lower concentrate than the instrument detection limit.

Abstract:L-tryptophan, one of the aromatic amino acids, is widely used in the fields of medicine, food and feed additives. The phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) plays an important role in glucose transport and phosphorylation in Escherichia coli. PTS-mediated regulation dominates the carbohydrates’ uptake and metabolism in E. coli. We constructed L-tryptophan-producing bacteria containing two typical PTS mutations (ptsHIcrr- glf-glk+ and ptsG-) by Red homologous recombination system, and studied 50 L jar fermenter using fed-batch fermentation. Both PTS system mutants had a great impact on the biomass (increasing 47.0% and 17.6%, respectively), L-tryptophan production (increasing 25.9% and 9.4%, respectively), glucose conversion rate (increasing 26.5% and 17.4%, respectively) and byproduct acetic acid generation (slightly increased and decreased，respectively).

Abstract:In order to produce hyaluronate lyase of high yield, we optimized the fermentation Arthrobacter globiformis A152 in quadruple fermentation of 5 L, and studied the kinetics of fermentation. Both the highest biomass and enzyme activity could be achieved when the rotation speed was 400 r/min and the ventilation volume was 3.5 L/min. In addition, digital models of cell growth, product synthesis and substrate consumption were built by equation of logistic, luedeking-piret, product synthesis and substrate consumption. Nonlinear fitting and estimation of optimal parameters were obtained by MATLAB. The model correlated well between prediction and experimental data, and reflected the change rules of cell growth, hyaluronidase synthesis and substrate consumption during the process of producing hyaluronate lyase. The establishment of fermentation kinetics digital models could provide basis for controlling and prediction of the production process.

Abstract:Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L.