Abstract:The emergence and spread of antimicrobial resistance has become a serious global issue. Bacterial characteristics, such as antimicrobial resistance genes, virulence-associated genes, plasmid types, and phylogenetic relationship among different strains, are the keys to unravel the occurrence and dissemination of antimicrobial resistance. However, the accuracy and efficiency of the traditional techniques, such as polymerase chain reaction and pulsed field gel electrophoresis is insufficient to underlying the mystery of antimicrobial resistance. Recently, the whole genome sequencing and high-throughput bioinformatics analysis have been successfully used in antimicrobial resistance studies, helping scientists to obtain the nature of antimicrobial resistance bacteria quickly, and more precisely to paint the evolutionary relationship among different strains. Therefore, in this study, we aim to systematically introduce the recent development of whole genome sequencing analysis, including different methods and corresponding characteristics of library preparation, platform sequencing, data analysis, and the latest application of the technology in the antimicrobial resistance research. We hope that this review can provide more comprehensive knowledge about whole genome sequencing and bioinformatic analysis for antimicrobial resistance research.

Abstract:Bacterial biofilm refers to a tunicate-like biological group composed of polysaccharide, protein and nucleic acid secreted by bacteria on the surface of the mucous membrane or biological material. The biofilm formation is a major cause of chronic infections. Bacteria could produce some secondary metabolites during the growth and reproduction. Some of them act as signaling molecules allowing bacteria to communicate and regulate many important physiological behaviors at multiple-cell level, such as bioluminescence, biofilm formation, motility and lifestyles. Usually, these signal molecules play an important role in the formation of bacterial biofilm. We review here the effects of related signal molecules of Quorum Sensing, cyclic diguanylate, Two-Component Systems and sRNA on the biofilm formation. Focusing on these regulation mechanism of signal molecules in the process of biofilm formation is necessary for the prevention and treatment of some chronic diseases.

Abstract:Ethyl carbamate (EC), a carcinogenic and teratogenic chemical that is widely distributed in various alcoholic beverages, has attracted much attention. Microbial enzymatic degradation of EC in rice wine is always efficient and attractive. In this review, we summarize the research progress and problems of microbial enzymatic elimination of EC in rice wine from three aspects: the mechanisms of EC formation in rice wine, the research progress of acid urease, and the research progress of urethanase. Then, we propose the corresponding strategies to solve the problems: screening new urethanase with satisfied enzyme properties, food-grade expression and directed evolution of the bifunctional Fe3+-dependent acid urease and acid urease used in combination with urethanase to eliminate both urea and EC in rice wine.

Abstract:Baculovirus expression vector system (BEVS) has been successfully applied to the over-expression of various proteins, thus providing sufficient materials for vaccine research. Compared to other systems, BEVS has many advantages: baculovirus solely being parasitic in invertebrates, the resultant products conferring high safety to mammalian, high expression level of recombinant proteins, preferable folding for eukaryotic protein, proper post-translational modification required for biological function, suitable for multiple genes co-expression and large-scale production with serum-free culture media. To better understand the advantages and prospective of BEVS for the vaccine research, this article will review the development of BEVS and its application on vaccine research.

Abstract:Sclerotinia sclerotiorum is a typical necrotrophic plant pathogenic fungus that distributes worldwide and causes severe diseases on a broad-range of plant species. Studies on S. sclerotiorum have been mainly focused on biology and pathology. The development of high-throughput technologies enabled multi-omics approaches for systems biology. This review summarizes current researches on S. sclerotiorum and proposes systemic strategies for understanding its biology and pathology, to provide novel insights and references for further investigation on molecular biology and pathogenesis of the pathogenic fungi and the pathosystems.

Abstract:Long non-coding RNAs (lncRNAs) are members of RNA that are structurally similar to mRNA. They cannot encode proteins because they do not have a conserved open reading frame. LncRNAs were once regarded as abnormalities or noises or without any biological function after gene transcription. With the further development of research, it has been found that it can participate in normal or abnormal biological processes as an important regulator. LncRNAs are closely related to the development of nervous system function, metabolic disorders and tumors. LncRNAs abnormally expressed in cervical cancer participate in the regulation of various biological processes of cervical cancer by inhibiting or promoting tumors. This article reviews the recent reports on the abnormal regulation, molecular regulation mechanism and potential clinical application of lncRNAs in cervical cancer.

Abstract:Isopeptide bond-mediated molecular superglue is the irreversible covalent bond spontaneously formed by the side chains of lysine (Lys) and asparagine/aspartic acid (Asn/Asp) residues. The peptide-peptide interaction is specific, stable, and can be achieved quickly without any particular physicochemical factor. In the light of recent progress by domestic and foreign researchers, here we summarize the origin, assembly system and mechanism of isopeptide bond reaction, as well as the molecular cyclization and protein topological structure mediated by it. The prospect for its application in synthetic vaccine, hydrogel and bacterial nanobiological reactor is further discussed.

Abstract:Glucoamylase is a critical ingredient for saccharification in the starch decomposition, and widely used in food, pharmaceutical and fermentation industries. Glucoamylases are usually thermostable and have peak activities at high temperature, as required for the industrial process of glucose production. In this study, a glucoamylase gene belonging to the glycoside hydrolase (GH) family 15, Tlga15A, was cloned from Talaromyces leycettanus JCM12802, and successfully expressed in Pichia pastoris GS115. Recombinant glucoamylase TlGA showed optimal activities at pH 4.5 and 75 °C. The result of thermostability analysis showed that TlGA retained above 70% activity after incubating for 1 h at 65 °C, and 43% residual activity after 30 min at 70 °C. Moreover, TlGA had high resistance to most metal ions and chemical reagents tested. Various starch substrates could be hydrolyzed by TlGA, including soluble starch (255.6±15.3) U/mg, amylopectin (342.3± 24.7) U/mg, glycogen (185.4±12.5) U/mg, dextrin (423.3±29.3) U/mg and pullulan (65.7±8.1) U/mg. The primary, secondary and tertiary structures of glucoamylase were further analyzed. The low ratio of Gly in the primary structure and low exposed nonpolarity solvent accessible surface in the tertiary structure may be the main reasons for TlGA’s thermostability. These results show that TlGA is great promising for potential use in the commercial production of glucose syrups. Moreover, this research will provide knowledge and innovating ideas for the improvement of glucoamylase thermostability.

Abstract:Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.

Abstract:Glutamate decarboxylase, a unique pyridoxal 5′-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T5015) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.

Abstract:Gibberellin is an essential plant hormone that plays an important regulatory role throughout the life cycle of higher plants. A total of 23 genes involved in gibberellin action were identified from Phyllostachys edulis genome, including 8 GA20ox and 1 GA3ox genes involved in the gibberellin biosynthesis, 8 GA2ox genes involved in the metabolism of gibberellin, 2 GID1 genes involved in gibberellin perception, 2 GID2 genes and 2 DELLA genes involved in gibberellin signal transduction. Phylogenetic analysis of these genes from Arabidopsis, Oryza sativa and Phyllostachys edulis revealed that gibberellin biosynthesis, metabolism, and signaling pathways are conserved in these species. Treatment of seeds and seedlings of bamboo with exogenous gibberellin revealed that gibberellin significantly increased seed germination rate and stem elongation of seedlings, and had the best concentration of action. The expression levels of GA20ox and GA3ox genes in the bamboo seedlings were down-regulated and the expression of the active gibberellin-degrading gene GA2ox was up-regulated after GA3 treatment, and the transcriptional level of the gibberellin receptor GID1 and the positive regulatory gene GID2 was significantly increased while the expression of the negative regulatory gene DELLA was decreased. These genes have significant differences in the expression of different spatial locations of bamboo shoot stems, GA20ox, GA3ox, GA2ox, GID1 and GID2 are all expressed in the upper part of bamboo shoots, while the repressor gene DELLA accumulates at the bottom of the shoots and is hardly expressed at the top.

Abstract:Cordyceps militaris exopolysaccharides (EPS) have many pharmacological activities such as boosting immunity and antifatigue. To obtain EPS efficiently, we added moderate Vernonia amygdalina leaf powder as inducer to the fermentation medium to promote the production of Cordyceps militaris EPS and studied the infrared absorption spectrum and antioxidant activities of the EPS after optimization. The optimum liquid fermentation conditions were as follows: addition of Vernonia amygdalina leaf powder of 8 g/L, fermentation duration of 9 d, initial pH of 6.5, inoculation quantity of 5.0 mL. Under such a condition, the yield of Cordyceps militaris EPS reached (5.24±0.28) mg/mL, increased by 205.20% compared to the control group without adding Vernonia amygdalina leaf powder. Results of infrared analysis and antioxidant activity showed that the Vernonia amygdalina leaves had little effect on the structure and activities of Cordyceps militaris EPS. The results of this research suggest that Vernonia amygdalina leaf can enhance the production of Cordyceps militaris EPS effectively, and provides a novel method for efficient production of EPS in Cordyceps militaris.

Abstract:Signal transducer and activator of transcription 3 (STAT3) and Chemokine CX3C ligand 1 (Fractalkine/CX3CL1) play important roles in vascular inflammation and injury. To study if STAT3 promotes vascular endothelial cell proliferation and migration through fractalkine, we overexpressed or knocked down STAT3 in vascular endothelial cells, and used quantitative real-time PCR and Western blotting to determine the effect of STAT3 on fractalkine expression. The wild type and STAT3 binding site mutant fractalkine promoter luciferase reporter plasmids were constructed, and luciferase activity assays were used to explore the effect of STAT3 on the transcriptional activity of the fractalkine promoter. MTT assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on the proliferation rate of vascular endothelial cells. Scratch assays were used to detect the effect of overexpression or knockdown of STAT3 or fractalkine on vascular endothelial cell migration. There results showed that overexpression of STAT3 could promote fractalkine expression, and knockdown of STAT3 could down-regulate fractalkine expression. STAT3 could directly bind to the promoter of fractalkine to promote its transcriptional activity via binding the GAS site of the fractalkine promoter. Knockdown of STAT3 could inhibit the migration of vascular endothelial cell, and overexpression of fractalkine antagonized this inhibition. Our data concluded that STAT3 promotes the proliferation and migration of vascular endothelial cell by binding the GAS site of the fractalkine promoter to promote fractalkine transcriptional activity and expression.

Abstract:In order to provide a theoretical basis for better understanding the function and properties of proteins, we proposed a simple and effective feature extraction method for protein sequences to determine the subcellular localization of proteins. First, we introduced sparse coding combined with the information of amino acid composition to extract the feature values of protein sequences. Then the multilayer pooling integration was performed according to different sizes of dictionaries. Finally, the extracted feature values were sent into the support vector machine to test the effectiveness of our model. The success rates in data set ZD98, CH317 and Gram1253 were 95.9%, 93.4% and 94.7%, respectively as verified by the Jackknife test. Experiments showed that our method based on multilayer sparse coding can remarkably improve the accuracy of the prediction of protein subcellular localization.

Abstract:Endogenous peptides, in the form of cytokines, growth hormones and hormone peptides, play an important role in human hormones, nerves, cell growth and reproduction. Neuropeptide is a kind of endogenous peptide, which is related to the physiological activities of pain, sleep, emotion, learning and memory. Neuropeptides exist not only in the nerve cells of the brain, but also in other body fluids and organs. At present, there is still a lack of research on endogenous peptides, especially on neuropeptides. In this study, high-throughput liquid chromatography tandem mass spectrometry was used to identify the distribution of endogenous peptides in the pancreas, heart, liver and kidney as well as the types of neuropeptides. The results showed that the number of endogenous peptides and neuropeptides in the liver was the highest while that of the pancreas was the lowest. The identified endogenous peptides were organ-specific and presented different dynamic distribution in four kinds of organs. The number of LPV (Longest peptide variant) of neuropeptide in the four organs varies greatly, and the distribution of gene family is also different. For example, neuropeptide in pancreas belongs to Glucagon family, while neuropeptide in heart belongs to ACBD7, Granins, PEBP and other families. The identification results will provide reference value for the mechanism study of diseases and the research and development of therapeutic drugs.

Abstract:To develop an enzyme-linked immunosorbent assay (ELISA)-based high throughput screening (HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta (DE3) competent cells were transformed with β-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography. The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mL β-catenin, with the Z￠ factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.

Abstract:Multi-epitope recombinant diagnostic antigen (designated ‘B102’) of Mycobacterium tuberculosis (Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens (PstS1, ESAT6, CFP10, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation, protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively. The McNemer analysis suggested there was no statistical difference between the ‘Gold standard’ and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.

Abstract:Yeast autolysis under solid-state fermentation can effectively promote the release of various active substances, thereby improving the quality of yeast products. The optimal process for yeast autolysis under solid-state fermentation was obtained by optimizing the autolysis temperature, autolysis time and the zinc ion concentration. We analyzed the indexes of free amino acid, soluble protein and α-amino nitrogen in the fermentation material, as well as A260/A280 ratio to determine yeast autolysis process conditions in the solid-state fermentation. On the basis of the obtained data, L9 (33) orthogonal test was designed to optimize the solid-state fermentation parameters for yeast autolysis: temperature at 40, 50 and 55 °C; time 12, 18 and 24 h; zinc ion concentration 2, 4 and 8 mg/kg. The optimum process conditions for yeast autolysis were: autolysis temperature 55 °C, time 18 h, zinc ion concentration 2 mg/kg, and soluble protein content reached 9.31 mg/g, free amino acid 14.36 mg/g, α-amino nitrogen 10.16 μg/g and A260/A280 1.73. After optimization of the process, the soluble protein, free amino acid and α-amino nitrogen contents of the yeast autolysis production can be significantly increased, thereby obviously improving the quality of the composite culture.