PhaGAA: an integrated web server platform for phage genome annotation and analysis.

MOTIVATION: Phage genome annotation plays a key role in the design of phage therapy. To date, there have been various genome annotation tools for phages, but most of these tools focus on mono-functional annotation and have complex operational processes. Accordingly, comprehensive and user-friendly platforms for phage genome annotation are needed. RESULTS: Here, we propose PhaGAA, an online integrated platform for phage genome annotation and analysis. By incorporating several annotation tools, PhaGAA is constructed to annotate the prophage genome at DNA and protein levels and provide the analytical results. Furthermore, PhaGAA could mine and annotate phage genomes from bacterial genome or metagenome. In summary, PhaGAA will be a useful resource for experimental biologists and help advance the phage synthetic biology in basic and application research. AVAILABILITY AND IMPLEMENTATION: PhaGAA is freely available at http://phage.xialab.info/.

[Purification, characterization and application of ε-poly-L. lysine- degrading enzyme from Streptomyces sp. M-Z18 ].

[OBJECTIVE] The ε-poly-L-lysine-degrading enzyme (Pld) derived from Streptomyces sp. M-Z18 was purified and characterized. Furthermore, Pld was used to produce the low polymerization of ε-poly-L-lysine (ε-PL). [METHODS] Pld was purified to electrophoretical homogeneity through HiTrapTM Butyl HP hydrophobic chromatography after pretreated by ultrasonic and NaSCN dissolving. Subsequently, enzymatic characteristics, kinetic parameters and the time profile of ε-PL degradation by the purified Pld were studied. Meanwhile, we examined the effect of ε-PL with different degrees of polymerization on the minimal inhibitory concentration of bacteria and fungi. [RESULTS] Pld was purified to homogeneity with a final fold of 80.4 and an overall yield of 59.3%. The optimal temperature and pH for the purified Pld were 370C and 7. 0, respectively. Moreover, the Km with L-lysyl-p-nitroanilide as substrate was calculated to be 0. 621 mmol/L, and the Vmax was 701. 16 nmol/min.mg. Pld was stable in the range of pH 7. 0 - 10. 0, and temperature up to 500 C, respectively. Time profile of ε-PL degradation by the purified Pld indicated that Pld catalyzed endo-type degradation of ε- PL. The experiments of minimal inhibitory showed that ε-PL with high degree of polymerization (30 - 35) had a superior antibacterial effect on bacteria and the low degree of polymerization ε-PL (8 -20) had a better antibacterial effect on yeasts. However, ε-PL with various degrees of polymerization had a poor antibacterial effect on mould. [ CONCLUSION] The present result showed that an endo-type Pld from ε-PL-producing strain was purified. Meanwhile, it is proved that ε-PL with different degrees of polymerization have exhibited significant different antibacterial effects on microorganism.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity. Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Insights into the role of glucose and glycerol as a mixed carbon source in the improvement of ε-poly-L-lysine productivity.

Using glucose-glycerol mixed carbon source has proved to be an effective strategy for ε-poly-L-lysine (ε-PL) production with rapid cell growth and much higher ε-PL productivity. In this study, we attempt to focus on key enzymes and intracellular energy cofactors to reveal the underlying mechanisms involved in such significant improvements. The activities of key enzymes involved in the pentose phosphate pathway, TCA cycle, anaplerotic pathway and the aspartate family amino acid biosynthesis pathway as well as ε-PL synthetase showed overall enhancement with the mixed carbon source, especially in the late stages of fermentation, compared with those in either glucose or glycerol single carbon sources. Moreover, the intracellular cofactors in terms of NADH and ATP kept higher formation and consumption rates in the mixed carbon source, respectively, throughout batch fermentation. As a result, Streptomyces sp. M-Z18 could be accelerated in cell growth and precursor L-lysine biosynthesis in the mixed carbon source, thus finally shortening fermentation time and enhancing ε-PL productivity. Understanding this process will provide information for the rational regulation of the metabolism network of the quantative production of ε-PL by metabolic engineering.

Rehmannia glutinosa polysaccharide induces maturation of murine bone marrow derived Dendritic cells (BMDCs).

Purified Rehmannia glutinosa polysaccharide (RGP) is used as functional foods for the prevention and treatment of various diseases. In this study, we examined the effects of RGP on phenotypic and functional maturation of murine bone marrow derived Dendritic cells (BMDCs). Phenotypic maturation of BMDCs was confirmed by conventional scanning electron microscopy (SEM), flow cytometry (FCM) and functional maturation by transmission electron microscopy (TEM), cytochemistry assay, Acid phosphatase (ACP) activity, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA).We found that RGP up-regulated the expression of CD40, CD80, CD83, CD86 and MHC II molecules of BMDCs, down-regulated pinocytosis and phagocytosis activity, induced IL-12 and TNF-α production of BMDCs. It is therefore concluded that RGP can effectively promote the maturation of DCs. Our study provides evidence and rationale on using RGP in various clinical conditions to enhance host immunity and suggests RGP as a potent adjuvant for the design of DC-based vaccines.

The expression and clinical significance of melanoma-associated antigen-A1, -A3 and -A11 in glioma.

Melanoma-associated antigens (MAGEs) were initially identified in melanoma and have since been widely studied. Melanoma-associated antigen-As (MAGE-As), a subfamily of MAGEs, are expressed in germ cells and various types of cancer, and are considered to be ideal targets for cancer immunotherapy. Glial cells and melanocytes originate from the neural ectoderm, so tumors derived from these two types of cells, i.e. gliomas and melanomas, may have common biological characteristics. However, studies on the expression of the MAGE-A family in gliomas are limited and conflicting. In the present study, the expression levels of MAGE-A1, -A3 and -A11 were detected by immunohistochemistry, and the association of their expression levels with the clinicopathological parameters, overall survival (OS) and ki-67 labeling indices of glioma patients were analyzed. The results showed that i) the expression levels of MAGE-A1, -A3 and -A11 proteins in the glioma tissues were 64.1, 51.3 and 57.7%, respectively and that no MAGE-A1, -A3 or -A11 expression was detected in the normal brain specimens; ii) the expression levels of MAGE-A1 and -A11 increased with ascending pathological grades and were positively correlated with the ki-67 labeling index; and iii) the OS of the patients in the groups with high MAGE-A1 (P=0.005) and -A11 (P=0.019) expression was statistically lower compared with the groups with low expression and no significant differences in OS were detected between the patients in the groups with high and low MAGE-A3 expression (P=0.304). Based on these results, we conclude that MAGE-A1, -A3 and -A11 may be used as ideal targets for glioma immunotherapy, and that MAGE-A1 and -A11 expression may be involved in tumor cell proliferation. These proteins may be potential indicators of a poor prognosis in glioma patients.