Dynamic changes in the gut microbiota after bismuth quadruple therapy and high-dose dual therapy for Helicobacter pylori eradication.

Background: A novel regimen with high-dose dual therapy (HDDT) has emerged, but its impact on the gut microbiota is not well understood. This study aimed to evaluate the impact of HDDT on the gut microbiota and compare it with that of bismuth quadruple therapy (BQT).

Methods: We enrolled outpatients (18-70 years) diagnosed with Helicobacter pylori infection by either histology or a positive C-urea breath test (C-UBT) and randomly assigned to either the BQT or HDDT group.

The Synthesis, Characterization and Anti-Tumor Activity of a Cu-MOF Based on Flavone-6,2'-dicarboxylic Acid.

A novel two-dimensional copper(II) framework (LDU-1), formulated as {[Cu(L)·2NMP}n (HL = flavone-6,2'-dicarboxylic acid, NMP = N-Methyl pyrrolidone), has been constructed under solvothermal conditions and characterized by single-crystal X-ray diffraction, infrared spectroscopy (IR), thermogravimetric analysis and powder X-ray diffraction (PXRD). In the crystal structure, the Cu(II) shows hex-coordinated with the classical Cu paddle-wheel coordination geometry, and the flavonoid ligand coordinates with the Cu(II) ion in a bidentate bridging mode. Of particular interest of LDU-1 is the presence of anti-tumor activity against three human cancer cell lines including lung adenocarcinoma(A549), Michigan cancer foundation-7 (MCF-7), erythroleukemia (K562) and murine melanoma B16F10, indicating synergistic enhancement effects between metal ions and organic linkers.

EppR, a new LysR-family transcription regulator, positively influences phenazine biosynthesis in the plant growth-promoting rhizobacterium Pseudomonas chlororaphis G05.

Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor.

LysR-type transcriptional regulator FinR is required for phenazine and pyrrolnitrin biosynthesis in biocontrol Pseudomonas chlororaphis strain G05.

Phenazine-1-carboxylic acid and pyrrolnitrin, the two secondary metabolites produced by Pseudomonas chlororaphis G05, serve as biocontrol agents that mainly contribute to the growth repression of several fungal phytopathogens. Although some regulators of phenazine-1-carboxylic acid biosynthesis have been identified, the regulatory pathway involving phenazine-1-carboxylic acid synthesis is not fully understood. We isolated a white conjugant G05W03 on X-Gal-containing LB agar during our screening of novel regulator candidates using transposon mutagenesis with a fusion mutant G05Δphz::lacZ as a recipient.

Pip serves as an intermediate in RpoS-modulated phz2 expression and pyocyanin production in Pseudomonas aeruginosa.

Pyocyanin, a main virulence factor that is produced by Pseudomonas aeruginosa, plays an important role in pathogen-host interaction during infection. Two copies of phenazine-biosynthetic operons on genome, phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2), contribute to phenazine biosynthesis. In our previous study, we found that RpoS positively regulates expression of the phz2 operon and pyocyanin biosynthesis in P.

Overexpression of phzM contributes to much more production of pyocyanin converted from phenazine-1-carboxylic acid in the absence of RpoS in Pseudomonas aeruginosa.

Pyocyanin produced by Pseudomonas aeruginosa is a key virulence factor that often causes heavy damages to airway and lung in patients. Conversion of phenazine-1-carboxylic acid to pyocyanin involves an extrametabolic pathway that contains two enzymes encoded, respectively, by phzM and phzS. In this study, with construction of the rpoS-deficient mutant, we first found that although phenazine production increased, pyocyanin produced in the mutant YTΔrpoS was fourfold much higher than that in the wild-type strain YT.

, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in G05.

In our previous study, pyrrolnitrin produced in G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05ΔΔ:: and S17-1 (pUT/mini-Tn5Kan).

phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.

Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA B C D E F G ) and phz2 (phzA B C D E F G ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria.

Pyrrolnitrin is more essential than phenazines for Pseudomonas chlororaphis G05 in its suppression of Fusarium graminearum.

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) disease in cereal crops worldwide. Infection with this fungal phytopathogen can regularly cause severe yield and quality losses and mycotoxin contamination in grains. In previous other studies, one research group reported that pyrrolnitrin had an ability to suppress of mycelial growth of F.

Reciprocal enhancement of gene expression between the phz and prn operon in Pseudomonas chlororaphis G05.

In previous studies with Pseudomonas chlororaphis G05, two operons (phzABCDEFG and prnABCD) were confirmed to respectively encode enzymes for biosynthesis of phenazine-1-carboxylic acid and pyrrolnitrin that mainly contributed to suppression of some fungal phytopathogens. Although some regulators were identified to govern their expression, it is not known how two operons coordinately interact. By constructing the phz- or/and prn- deletion mutants, we found that in comparison with the wild-type strain G05, phenazine-1-carboxylic acid production in the mutant G05Δprn obviously decreased in GA broth in the absence of prn, and pyrrolnitrin production in the mutant G05Δphz remarkably declined in the absence of phz.

Construction of a β-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in Pseudomonas chlororaphis G05.

In our recent work, we found that pyrrolnitrin, and not phenazines, contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain.

[Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1].

Unlabelled: Pyocyanin, an important virulence factor, is synthesized and secreted by Pseudomonas aeruginosa PAO1and plays a critical role in pathogen-host interaction during infection. Sigma38 (σ38, σS) is a central regulator for many virulence production in pathogens.

Objective: Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma38 factor in Pseudomonas aeruginosa PAO1.

Enhanced expression of GCRV VP6 in CIK cells by relative sequence optimization.

Efficient expression of target protein is one of strategies for gene therapy or vaccine design. Many studies showed that codon optimization could enhance the expression of target proteins. In this paper, a target sequence of about 1.

[Positive regulation in expression of the phenazine-producing operon phz2 mediated by pip in Pseudomonas aeruginosa PAO1].

Unlabelled: Pseudomonas aeruginosa PAO1, an opportunistic pathogenic bacterium, produces phenazine and its derivatives which play a critical role in pathogen-host interaction during its infection. In a biological control strain P. chlororaphis PCL1391, Pip positively regulates PCN production.

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors.

Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein.

[Differential expression of two phenazine-producing loci mediated by deficiency of the global regulator rsmA in Psedomonas aeruginosa PAO1].

Unlabelled: In many Pseudomonas, RsmA mediates the production of a set of secondary metabolites or virulence factors.

Objective: Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1.

Methods: We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA.

[Trans-splicing of Cys mutated coagulation factor VIII].

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA.

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors.

Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo.

[Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system].

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system.

Optimization of exopolysaccharides production from a novel strain of Ganoderma lucidum CAU5501 in submerged culture.

This study aimed at optimizing the medium of a new Ganoderma lucidum strain CAU5501 to enhance the yield of exopolysaccharides (EPS) and mycelial growth. Firstly, the suitable level of glucose, magnesium, phosphate and C/N ratio was determined by single factor experiment. Subsequently, the optimum concentrations of these medium components were investigated using the orthogonal matrix method.

[Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells].

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed.

[Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function].

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein.

[Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII].

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity.

[Post-translational ligation and function of dual-vector transferred split CFTR gene].

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel.