Whole-genome sequencing and analysis of Chryseobacterium arthrosphaerae from Rana nigromaculata.

Chryseobacterium arthrosphaerae strain FS91703 was isolated from Rana nigromaculata in our previous study. To investigate the genomic characteristics, pathogenicity-related genes, antimicrobial resistance, and phylogenetic relationship of this strain, PacBio RS II and Illumina HiSeq 2000 platforms were used for the whole genome sequencing. The genome size of strain FS91703 was 5,435,691 bp and GC content was 37.78%. A total of 4,951 coding genes were predicted; 99 potential virulence factors homologs were identified. Analysis of antibiotic resistance genes revealed that strain FS91703 harbored 10 antibiotic resistance genes in 6 categories and 2 multidrug-resistant efflux pump genes, including adeG and farA. Strain FS91703 was sensitive to ß-lactam combination drugs, cephem, monobactam and carbapenems, intermediately resistant to phenicol, and resistant to penicillin, aminoglycosides, tetracycline, fluoroquinolones, and folate pathway inhibitors. Phylogenetic analysis revealed that strain FS91703 and C. arthrosphaerae CC-VM-7T were on the same branch of the phylogenetic tree based on 16 S rRNA; the ANI value between them was 96.99%; and the DDH values were 80.2, 72.2 and 81.6% by three default calculation formulae. These results suggested that strain FS91703 was a species of C. arthrosphaerae. Pan-genome analysis showed FS91703 had 566 unique genes compared with 13 other C. arthrosphaerae strains, and had a distant phylogenetic relationship with the other C. arthrosphaerae strains of the same branch in phylogenetic tree based on orthologous genes. The results of this study suggest that strain FS91703 is a multidrug-resistant and highly virulent bacterium, that differs from other C. arthrosphaerae strains at the genomic level. The knowledge about the genomic characteristics and antimicrobial resistance of strain FS91703 provides valuable insights into this rare species, as well as guidance for the treatment of the disease caused by FS91703 in Rana nigromaculata.

Evolution of Rheological Behaviors of Styrene-Butadiene-Styrene/Crumb Rubber Composite Modified Bitumen after Different Long-Term Aging Processes.

In this study, a new type of composite modified bitumen was developed by blending styrene-butadiene-styrene (SBS) and crumb rubber (CR) with a chemical method to satisfy the durability requirements of waterproofing material in the waterproofing layer of high-speed railway subgrade. A pressure-aging-vessel test for 20, 40 and 80 h were conducted to obtain bitumen samples in different long-term aging conditions. Multiple stress creep recovery (MSCR) tests, linear amplitude scanning tests and bending beam rheometer tests were conducted on three kinds of asphalt binders (SBS modified asphalt, CR modified asphalt and SBS/CR composite modified asphalt) after different long-term aging processes, including high temperature permanent deformation performance, resistance to low temperature thermal and fatigue crack. Meanwhile, aging sensitivities were compared by different rheological indices. Results showed that SBS/CR composite modified asphalt possessed the best properties before and after aging. The elastic property of CR in SBS/CR composite modified asphalt improved the ability to resist low temperature thermal and fatigue cracks at a range of low and middle temperatures. Simultaneously, the copolymer network of SBS and CR significantly improved the elastic response of the asphalt SBS/CR modified asphalt at a range of high temperatures. Furthermore, all test results indicated that the SBS/CR modified asphalt possesses the outstanding ability to anti-aging. SBS/CR is an ideal kind of asphalt to satisfy the demand of 60 years of service life in the subgrade of high speed railway.

Stimulus-Responsive Plasmonic Chiral Signals of Gold Nanorods Organized on DNA Origami.

In response to environmental variations, living cells need to arrange the conformational changes of macromolecules to achieve the specific biofunctions. Inspired by natural molecular machines, artificial macromolecular assemblies with controllable nanostructures and environmentally responsive functions can be designed. By assembling macromolecular nanostructures with noble metal nanoparticles, environmental information could be significantly amplified and modulated. However, manufacturing dynamic plasmonic nanostructures that are efficiently responsive to different stimuli is still a challenging task. Here we demonstrate a stimulus-responsive plasmonic nanosystem based on DNA origami-organized gold nanorods (GNRs). L-shaped GNR dimers were assembled on rhombus-shaped DNA origami templates. The geometry and chiral signals of the GNR nanoarchitectures respond to multiple stimuli, including glutathione reduction, restriction enzyme action, pH change, or photoirradiation. While the glutathione reduction or restriction enzyme caused irreversible changes in the plasmonic circular dichroism (CD) signals, both pH and light irradiation triggered reversible changes in the plasmonic CD. Our system transduces external stimuli into conformational changes and circular dichroism responses in near-infrared (NIR) wavelengths. By this approach, programmable optical reporters for essential biological signals can be fabricated.

A novel hedgehog inhibitor iG2 suppresses tumorigenesis by impairing self-renewal in human bladder cancer.

Tumor recurrence is still a major challenge for clinical treatment of bladder cancer. Cumulative evidences indicate cancer stem cells (CSCs) contribute to drug resistance and leave a putative source for disease relapse. Identifying novel agents targeting CSCs may represent a new paradigm in the therapy of bladder cancer. Here, we separated a novel hedgehog (Hh) inhibitor, iG2, from streptomyces roseofulvus, which dramatically blocked the activation of Gli2 in bladder cancer cells. The iG2 strongly repressed the growth of cancer cells rather than the peri-tumor stroma cells. Attenuated proliferation and enhanced apoptosis of tumor cells were observed upon iG2 stimulation. Furthermore, iG2 reduced the self-renewal ability of bladder CSCs as well as the tumor formation. Collectively, iG2 is potentially used as a novel therapeutic agent for bladder cancer by targeting self-renewal through inhibiting Hh pathway.

A Self-Assembled DNA Origami-Gold Nanorod Complex for Cancer Theranostics.

A self-assembled DNA origami (DO)-gold nanorod (GNR) complex, which is a dual-functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO-GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two-photon luminescence imaging. Particularly, the triangle shaped DO-GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO-GNR complex by two-photon laser or NIR laser irradiation. Moreover, the DO-GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO-GNR complex can achieve optimal two-photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo.

[Advances in the molecular mechanism of natural bacterial transformation--a review].

Naturally transformable bacteria are able to take up DNA to acquire new genetic traits in the environment. To be naturally transformed, bacteria need to establish a physiological state, called natural competence, in which DNA uptake and processing genes are expressed. DNA uptake proteins assemble a complex to pull exogenous DNA into the cytoplasm where it can recombine with the genome DNA or establish as a plasmid. In general, DNA uptake of bacteria could be divided into two stages: DNA is transported from the milieu to the periplasm at the first stage (for Gram-negative bacteria) and is translocated across the inner membrane at the second stage. Our work and other studies revealed new plasmid DNA transformation modes in Escherichia coli. Here, we first reviewed recent advances in the molecular mechanism of natural transformation and then described the distinctive plasmid transformation mode in E. coli.

Elucidation of Piericidin A1 biosynthetic locus revealed a thioesterase-dependent mechanism of α-pyridone ring formation.

Piericidins are a class of α-pyridone antibiotics that inhibit mitochondrial respiratory chain and exhibit antimicrobial, antifungal, and antitumor activities. Sequential analysis of Streptomyces piomogeues var. Hangzhouwanensis genome revealed six modular polyketide synthases, an amidotransferase, two methyltransferases, and a monooxygenase for piericidin A1 production. Gene functional analysis and deletion results provide overview of the biosynthesis pathway. Furthermore, in vitro characterization of the terminal polyketide synthase module with the thioesterase domain using ß-ketoacyl substrates was performed. That revealed a pathway where the α-pyridone ring formation is dependent on hydrolysis of the product ß, δ-diketo carboxylic acid by the C-terminal thioesterase followed by amidation and cyclization. These findings set the stage to investigate unusual enzymatic mechanisms in α-pyridone antibiotics biosynthesis, provide a foundation for genome mining of α-pyridone antibiotics, and produce analogs by molecular engineering.

[Effects of irrigation with regenerated water on the growth and quality of alfalfa].

A pot experiment was conducted to study the effects of irrigation with regenerated water on the growth, nutrient uptake, and quality of alfalfa. Four treatments were installed, i.e., irrigation with fresh water (Q), irrigation with regenerated water (Z), mixed irrigation with regenerated water and fresh water (Z + Q), and rotational irrigation with regenerated water and fresh water (Z/Q). Comparing with treatment Q, treatment Z increased the stem length, lateral branch number, and plant biomass significantly, but decreased leaf area. Treatments Z, Z + Q, and Z/Q increased plant soluble protein content by 78.43%, 83.68% and 72.53%, respectively, but treatment Z decreased plant soluble sugar content. Treatment Z increased plant Ca and Mg contents by 27.78% and 26.61%, and treatments Z and Z + Q decreased plant Fe content by 28.71% and 10.09%, respectively. Treatments Z, Z + Q, and Z/Q increased the cadmium content in above-ground part of alfalfa by 98.6%, 89.5%, and 59.0%, respectively, but the cadmium and lead contents were still lower than the thresholds of National Sanitation Standard (GB 13078-2001). It was suggested that regenerated water could be used as a source of irrigation water in alfalfa growth, but its long-term effect should be concerned.

Mutation screening of HSF4 in 150 age-related cataract patients.

PURPOSE: Heat shock transcription factor 4 (HSF4) regulates the expression of several heat shock protein (HSP) genes. HSPs are one of the major components responsible for lens protein organization. Recently, we found that mutations of HSF4 result in hereditary cataract. In this study, we explore the role of HSF4 in the development of age-related cataract. METHODS: We screened sequence variants of HSF4 in age-related cataract patients and the natural population from Shanghai, China. RESULTS: In individuals of natural populations, we detected no single nucleotide polymorphism (SNP) with a frequency higher than 5% in a complete coding region or in their exon-intron boundaries. In 150 age-related cataract patients, we identified seven sequence changes. We found an intronic G-->A transition (c.1020-25G>A) in one patient, a missense mutation (c.1078A>G) in exon 4 in two patients, a silent mutation (c.1223 C>T) in exon 5 in two patients, an intronic C-->T transition (c.1256+25C>T) in one patient, and a silent mutation in exon 6 (c.1286 C>T) in one patient. These five variants were not represented in 220 control individuals. We also identified an intronic C-->T transition (c.1019+9C>T) and a missense mutation (c.1243G>A) in exon 3 in three patients, but these two variants were also present in 100 control subjects. CONCLUSIONS: We identified five new HSF4 mutations in 150 age-related cataract patients, enlarging the spectrum of HSF4 mutations in cataract patients. This result indicates that HSF4 mutations account for only a small fraction of age-related cataracts.

[Apoptosis of HL-60 cells induced by crystal proteins from Bacillus thuringiensis Bt9875].

OBJECTIVE: To study the effect of Bt9875 crystal protein treated with proteinase K on human cancer cells, HL-60. METHODS: We used the methods of Thiazolyl Blue Tetrazolium Bromide, fluorescence microscopy examination, agarose gel electrophores and flow cytometry to detect the growth inhibition rate and apoptosis characters of the HL-60 cells that were treated with different concentration of Bt9875 crystal protein. RESULTS: Bt9875 crystal protein inhibited the growth of HL-60 cells evidently in a dose-dependent manner, with minimal effects on normal human peripheral blood mononuclear cells (PBMCs). The nuclei of HL-60 cells showed the characteristics of apoptosis. The analysis by flow cytometry indicated that the apoptosis rate of HL-60 cells was 52% after treatment with Bt9875 crystal protein (100 microg/mL). DNA analyzed by agarose gel electrophoresis showed "ladder" pattern. CONCLUSION: Bt9875 crystal protein could inhibit the growth of HL-60 and induced its apoptosis, which provided a foundation for use of Bt9875 crystal protein to cure the acute myeloid leukemia.

Effect of hypoxia on RAW264.7 macrophages apoptosis and signaling.

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. In this study, we have investigated cellular and molecular responses of murine macrophage-like RAW264.7 cells to low oxygen tension. Our study revealed that hypoxia induced RAW264.7 cells apoptosis and cell cycle arrest at G0/G1 phase. The result of Western blotting showed that the expression of apoptosis related signaling molecules, such as AKT and JNK was activated under hypoxia. The result of electrophoretic mobility shift assay showed that the DNA binding activity of nuclear factor kappa B (NF-kappaB) was also increased by hypoxic stimulation. Furthermore, gene expression profiles of RAW264.7 macrophages induced by hypoxia showed that hypoxia treatment may alter expression of genes related to apoptosis, survival, cell cycle, metabolism and structural matrix.

Hypoxia induces the activation of human hepatic stellate cells LX-2 through TGF-beta signaling pathway.

Hypoxia is a common environmental stress factor and is also associated with various physiological and pathological conditions such as fibrogenesis. The activation of hepatic stellate cells (HSCs) is the key event in the liver fibrogenesis. In this study, the behavior of human HSCs LX-2 in low oxygen tension (1% O2) was analyzed. Upon hypoxia, the expression of HIF-1alpha and VEGF gene was induced. The result of Western blotting showed that the expression of alpha-SMA was increased by hypoxic stimulation. Furthermore, the expression of MMP-2 and TIMP-1 genes was increased. Hypoxia also elevated the protein expression of the collagen type I in LX-2 cells. The analysis of TGF-beta/Smad signaling pathway showed that hypoxia potentiated the expression of TGF-beta1 and the phosphorylation status of Smad2. Gene expression profiles of LX-2 cells induced by hypoxia were obtained by using cDNA microarray technique.

Effects of rhDecorin on TGF-beta1 induced human hepatic stellate cells LX-2 activation.

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor beta (TGF-beta), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-beta, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-beta1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-beta1. Furthermore, the protein expression of smooth muscle-alpha-actin (alpha-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-beta1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.

Effect of WeiJia on carbon tetrachloride induced chronic liver injury.

AIM: To study the effect of WeiJia on chronic liver injury using carbon tetrachloride (CCl(4)) induced liver injury animal model. METHODS: Wistar rats weighing 180-220g were randomly divided into three groups: normal control group (Group A), CCl(4) induced liver injury control group (Group B) and CCl(4) induction with WeiJia treatment group (Group C). Each group consisted of 14 rats. Liver damage and fibrosis was induced by subcutaneous injection with 40% CCl(4) in olive oil at 3 mL/kg body weight twice a week for eight weeks for Groups B and C rats whereas olive oil was used for Group A rats. Starting from the third week, Group C rats also received daily intraperitoneal injection of WeiJia at a dose of 1.25 microg/kg body weight. Animals were sacrificed at the fifth week (4 male, 3 female), and eighth week (4 male, 3 female) respectively. Degree of fibrosis were measured and serological markers for liver fibrosis and function including hyaluronic acid (HA), type IV collagen (CIV), gamma-glutamyl transferase (gamma-GT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Alpha smooth muscle actin (alpha-SMA) and proliferating cell nuclear antigen (PCNA) immunohistochemistry were also performed. RESULTS: CCl(4) induction led to the damage of liver and development of fibrosis in Group B and Group C rats when compared to Group A rats. The treatment of WeiJia in Group C rats could reduce the fibrosis condition significantly compared to Group B rats. The effect could be observed after three weeks of treatment and was more obvious after eight weeks of treatment. Serum HA, CIV, ALT, AST and gamma-GT levels after eight weeks of treatment for Group C rats were 58+/-22 microg/L (P<0.01), 57+/-21 microg/L (P<0.01), 47+/-10 U/L (P<0.01), 139+/-13 U/L (P<0.05) and 52+/-21 U/L (P>0.05) respectively, similar to normal control group (Group A), but significantly different from CCl(4) induced liver injury control group (Group B). An increase in PCNA and decrease in alpha-SMA expression level was also observed. CONCLUSION: WeiJia could improve liver function and reduce liver fibrosis which might be through the inhibition of stellate cell activity.

Mutant DNA-binding domain of HSF4 is associated with autosomal dominant lamellar and Marner cataract.

Congenital cataracts cause 10-30% of all blindness in children, with one-third of cases estimated to have a genetic cause. Lamellar cataract is the most common type of infantile cataract. We carried out whole-genome linkage analysis of Chinese individuals with lamellar cataract, and found that the disorder is associated with inheritance of a 5.11-cM locus on chromosome 16. This locus coincides with one previously described for Marner cataract. We screened individuals of three Chinese families for mutations in HSF4 (a gene at this locus that encodes heat-shock transcription factor 4) and discovered that in each family, a distinct missense mutation, predicted to affect the DNA-binding domain of the protein, segregates with the disorder. We also discovered an association between a missense mutation and Marner cataract in an extensive Danish family. We suggest that HSF4 is critical to lens development.