Promoter engineering strategies for the overproduction of valuable metabolites in microbes.

Promoter engineering is an enabling technology in metabolic engineering and synthetic biology. As an indispensable part of synthetic biology, the promoter is a key factor in regulating genetic circuits and in coordinating multi-gene biosynthetic pathways. In this review, we summarized the recent progresses in promoter engineering in microbes. Specifically, the endogenous promoters are firstly discussed, followed by the statement of the influence of nucleotides exchange on the strength of promoters explored by site-selective mutagenesis. We then introduced the promoter libraries with a wide range of strength, which are constructed focusing on core promoter regions and upstream activating sequences by rational designs. Finally, the application of promoter libraries in the optimization of multi-gene metabolic pathways for high-yield production of metabolites was illustrated with a couple of recent examples.

[Characterization and secreted expression of dengue virus type I-IV envelope glycoprotein domain III in Pichia pastoris].

OBJECTIVE: To achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris. METHODS: EDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification. RESULTS: The recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera. CONCLUSION: DENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.

Integrated gene expression profile predicts prognosis of breast cancer patients.

Gene expression data has in recent years demonstrated the superior capacity to predict the prognosis of breast cancer patients unreceiving adjuvant chemotherapy comparing to the information available from traditional clinical and pathological sources. Meanwhile, adjuvant chemotherapy can significantly improve survival of breast cancer. It would be inappropriate to ignore its effect on prognosis. We hypothesized that an integrated gene expression profile can predict the prognosis of breast cancer patients receiving chemotherapy. Therefore, we screened the specific gene markers and constructed an integrated 24-gene signature by low-density microarray including the "poor signature" and genes related to resistance to chemotherapy. The gene signature stratified correctly patients into good prognosis group and poor prognosis group. In addition, the Kaplan-Meier analyses for disease-free survival as a function of the 24-gene signature showed highly significant differences between the two groups (Log Rank test P < 0.0001 = Univariate and multivariate Cox's proportional-hazards regression analyses indicated that the signature represents the strongest independent prognostic factor for breast cancer patients. When compared with single signature, such as Oncotype DX and 70 poor signature, the integrated signature showed more predominant power of predication in breast cancer patients receiving chemotherapy. Such integrated signature will critically aid clinical decision making at the level of individualization for most breast cancer patients receiving chemotherapy.

Use of serial analysis of gene expression to reveal the specific regulation of gene expression profile in asthmatic rats treated by acupuncture.

BACKGROUND: Asthma has become an important public health issue and approximately 300 million people have suffered from the disease worldwide. Nowadays, the use of acupuncture in asthma is increasing. This study intended to systematically analyze and compare the gene expression profiles between the asthmatic and acupuncture-treated asthmatic rat lung, and tried to gain insight into the molecular mechanism underlying the early airway response (EAR) phase of asthma treated by acupuncture. METHODS: Four tag libraries of serial analysis of gene expression (SAGE) were established from lung tissues of control rats (CK), asthmatic rats (AS), asthmatic rats treated by acupuncture (ASAC), and control rats treated by acupuncture (CKAC). Bioinformatic analyses were carried out by using the methods including unsupervised hierarchical clustering, functional annotation tool of the database for annotation, visualization, and integrated discovery (DAVID), gene ontology (GO) tree machine, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. RESULTS: There were totally 186 differentially expressed tags (P < 0.05, P(CK/AS)) between the libraries of CK and AS, 130 differentially expressed tags between libraries of AS/ASAC (P < 0.05, P(AS/ASAC)), and 144 differentially expressed tags between libraries of CK/CKAC (P < 0.05, P(CK/CKAC)). The gene expression profiles of AS and ASAC were more similar than other libraries via unsupervised SAGE clustering. By comparison of P(CK/AS) and P(AS/ASAC), the DAVID genes functional classification was found to be changed from "immune response" to "response to steroid hormone stimulus", and the GO term "antigen processing and presentation of peptide antigen" disappeared in P(AS/ASAC). Totally 3 same KEGG pathways were found among the three groups. Moreover, 21 specific tags of the acupuncture in treating asthma were detected using Venn diagrams. CONCLUSION: Our SAGE research indicates that the gene expression profile of the EAR phase of asthma could be effectively and specifically regulated by acupuncture, which suggests that the gene expression of immune response and steroid hormone may play an important role in the treatment.

Serial analysis of gene expression in a rat lung model of asthma.

BACKGROUND AND OBJECTIVE: The pathogenesis and molecular mechanism underlying asthma remain undetermined. The purpose of this study was to identify genes and pathways involved in the early airway response (EAR) phase of asthma by using serial analysis of gene expression (SAGE). METHODS: Two SAGE tag libraries of lung tissues derived from a rat model of asthma and controls were generated. Bioinformatic analyses were carried out using the Database for Annotation, Visualization and IntegratedDiscovery Functional Annotation Tool, Gene Ontology (GO) TreeMachine and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. RESULTS: A total of 26 552 SAGE tags of asthmatic rat lung were obtained, of which 12 221 were unique tags. Of the unique tags, 55.5% were matched with known genes. By comparison of the two libraries, 186 differentially expressed tags (P < 0.05) were identified, of which 103 were upregulated and 83 were downregulated. Using the bioinformatic tools these genes were classified into 23 functional groups, 15 KEGG pathways and 37 enriched GO categories. CONCLUSIONS: The bioinformatic analyses of gene distribution, enriched categories and the involvement of specific pathways in the SAGE libraries have provided information on regulatory networks of the EAR phase of asthma. Analyses of the regulated genes of interest may inform new hypotheses, increase our understanding of the disease and provide a foundation for future research.

Clinical Outcomes of Wulingsan Subtraction Decoction Treatment of Postoperative Brain Edema and Fever as a Complication of Glioma Neurosurgery.

Objective. To evaluate the efficacy of Wulingsan subtraction ( WLSS) decoction in the treatment of postoperative brain edema and fever as a complication of glioma neurosurgery. Methods. This retrospective study was conducted at the Department of Neurosurgery of Liaocheng People's Hospital. Patients hospitalized between March 2011 and December 2014 were divided into three groups: Group A received WLSS oral liquid (50 mL), twice a day; Group B received an intravenous infusion of mannitol; and Group C received WLSS combined with mannitol (n = 30 patients per group). All patients were treated for 10 days continuously. Therapeutic efficacy was evaluated by measuring body temperature and indicators of renal function before and 3, 5, and 10 days after treatment. Results. Compared to the other two groups, significantly greater clinical efficacy was observed in the patients treated with mannitol (Group B; P < 0.05), although marked clinical efficacy was also observed over time in patients treated with WLSS (Group A). After 5 days, the quantifiable effects of the WLSS and mannitol combination group (Group C) were substantial (P < 0.05). The renal damage in Group B was more obvious after 5 days and 10 days. Conclusion. Compared with mannitol treatment alone, WLSS combined with mannitol induced a more rapid reduction in body temperature. Our findings suggest that patients should be started on mannitol for 3 days and then switched to WLSS to achieve obvious antipyretic effects and protect renal function. This method of treatment should be considered for clinical applications.

Effect of total flavonoids from Scutellaria baicalensis on dopaminergic neurons in the substantia nigra.

The aim of the present study was to investigate the effect of Scutellaria baicalensis stem-leaf total flavonoid (SSTF) on the dopaminergic neurons in the substantia nigra in a mouse model of Parkinson's disease (PD). The mouse model was established by intravenous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). SSTF (5 mg/kg) was administered to the mice before or after MPTP injection, and the effects of SSTF on the behavior of the mice and the dopaminergic neurons in the substantia nigra were assessed. In addition, the level of serum malondialdehyde (MDA) was measured. Following injection of MPTP, the number of dopaminergic neurons in the substantia nigra was decreased and the neurons appeared atrophic. In addition, the level of serum MDA in the MPTP mice increased. The mean behavioral scores and the number of dopaminergic neurons in the SSTF treatment groups were significantly higher than in the MPTP group (P<0.05), and the mean serum MDA levels were significantly lower (P<0.05). Thus, SSTF improves the behaviors and the numbers of dopaminergic neurons in the substantia nigra in MPTP-induced PD in mice. These beneficial effects appear to be associated with the reduction in serum MDA.

[Sequencing and analysis of Vibrio cholerae typing phage VP3 genome].

DNA sequence and the genome of phage VP3 (a typing phage of V. cholera) were analyzed. A random library of VP3 DNA was constructed by shot-gun library method. The VP3 genome sequence was assembled with contigs sequences, the gaps between different contigs were filled with sequencing data from primer walking. ORFs were predicted; Phylogeny of DNA polymerase sequences was analyzed to determine the class of VP3; The activity of putative promoter genes were analyzed using lacZ report system. VP3 genome is a 39504bp of circular double-stranded DNA. Twenty-seven out of forty-nine putative ORFs were annotated; twenty gene products were homologous with T7-like phages, including DNAP, DNA replicative protein, capsid, tail tubular, tail fiber protein, and DNA packaged protein. The activity of the putative promoter regions was confirmed through cloning those regions to LacZ-fuse plasmid pRS1274 and analysis of the expression of beta galactosidase. The complete genomic sequence of VP3 and phylogenetic tree analysis suggests VP3 is a member of T7 phage family.

[Pharmacognostic studies on Desmodium gyrans].

OBJECTIVE: To study the characteristic features of Desmodium gyrans in order to provide a basis for rational exploitation and utilization of the herb. METHOD: Samples of the title plant were collected, the microscopic features of cross sections and powders were studied. TLC profiles and UV absorption of the plant extract were examined. RESULT: Calcium oxalate crystals were found in cells of transverse sections. Nonglandular hairs were observed on leaf surfaces. Characteristic peaks in the UV spectrum were identified. CONCLUSION: The distinct characteristic features revealed in this studies can serve as evidence for the identification of D. gyrans.

Protective Effect of Polysaccharides from Inonotus obliquus on Streptozotocin-Induced Diabetic Symptoms and Their Potential Mechanisms in Rats.

The present study aimed to evaluate the therapeutic effects of polysaccharides from Inonotus obliquus (PIO) on streptozotocin- (STZ-) induced diabetic symptoms and their potential mechanisms. The effect of PIO on body weight, blood glucose, damaged pancreatic ß-cells, oxidative stresses, proinflammatory cytokines, and glucose metabolizing enzymes in liver was studied. The results show that administration of PIO can restore abnormal oxidative indices near normal levels. The STZ-damaged pancreatic ß-cells of the rats were partly recovered gradually after the mice were administered with PIO 6 weeks later. Therefore, we may assume that PIO is effective in the protection of STZ-induced diabetic rats and PIO may be of use as antihyperglycemic agent.

Effects of acupuncture on the gene expression profile of lung tissue from normal rats.

Acupuncture has been demonstrated to be an effective treatment for various diseases. However, little attention has been paid to its physiological influences, especially on the changes in protein and mRNA levels following acupuncture treatment under normal conditions. In this study, we investigated the gene expression profile of lung tissue from acupuncture-treated normal rats and attempted to characterize the underlying mechanisms of the changes in expression. Three common acupoints, Dazhui (GV14), fengmen (BL12) and feishu (BL13) were selected for analysis, and 2 serial analyses of gene expression (SAGE) tag libraries of the lung tissues that were derived from the normal and acupuncture-treated rats were established. Bioinformatic analyses were carried out using the functional annotation tools of the database for annotation, visualization and integrated discovery (DAVID), the gene ontology (GO) Tree Machine and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. In total, 144 tags were differentially expressed (P<0.05), and the DAVID functional classification of genes demonstrated that the genes were divided into 6 types. Furthermore, GO Tree Machine analysis of the gene categories indicated that 10 enriched GO categories had become enriched after acupuncture, and that 15 KEGG pathways matched the differentially expressed tags of the 2 SAGE libraries. Our results show that the essential effects of acupuncture on normal rats include the regulation of macromolecular biosynthesis, transportation and metabolism. Cellular biosynthesis and cellular lipid metabolism are the common biological processes that occur in response to acupuncture under normal and morbid conditions, which may be the general physiological effects of acupuncture.