Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: De novo sequencing and analysis of tandem mass spectrometry data

Eight intact antimicrobial peptides were identified from the skin of Odorrana jingdongensis by de novo sequencing following low energy ESI CID Q-TOF MS/MS in positive-mode with the help of Edman degradation and structural similarity analysis. We devised exact mass measurements to discriminate the K/Q amino acid residue in the peptides between 2.0kDa to 3.8kDa. Moreover, the cleavage at the CS bond at the side chain of Met was observed in all the spectra of the peptides containing Met residue. And we found unusual cleavages within the intramolecular disulfide loop with high frequency. Our data revealed that the cleavage pathways are significantly different from those reported previously which are similar to the cycle peptide cleavage mode followed by the secondary cleavage at the CS bond on oxidized Cys. Thus, our results highly suggest that ion series generated from the cleavages within the intramolecular disulfide loop should be considered in both the top-down sequencing and the disulfide bridge location with the presence of a relatively high intensity of MH(+)-28 ion marker. Furthermore, our activity data implied that different AMPs may use different strategies to kill microbes.     

An improved sampling protocol for analysis of intracellular metabolites in Mortierella alpina

Sampling of intracellular metabolites in Mortierella alpina was investigated as part of a metabolomics study. After comparison of four sampling protocols, rapid filtration of the culture using a laboratory-made nylon filter and absorbent gauze under normal pressure followed by quenching in liquid N₂ and grinding (the improved protocol) was the most effective. Rapid filtration under normal pressure decreased intracellular metabolites leakage and subsequent grinding of cells contributed to intracellular metabolites extraction. The above quenching method together with 75?% (v/v) ethanol, buffered with 60?mM HEPES, at 80?°C for 3?min is therefore suitable for sampling intracellular metabolites in M. alpina.     

Increased intratumoral IL-22-producing CD4+ T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival

IL-22-producing CD4⁺ T cells (IL-22⁺CD4⁺ T cells) and Th22 cells (IL-22⁺IL-17^?IFN-γ^?CD4⁺ T cells) represent newly discovered T-cell subsets, but their nature, regulation, and clinical relevance in gastric cancer (GC) are presently unknown. In our study, the frequency of IL-22⁺CD4⁺ T cells in tumor tissues from 76 GC patients was significantly higher than that in tumor-draining lymph nodes, non-tumor, and peritumoral tissues. Most intratumoral IL-22⁺CD4⁺ T cells co-expressed IL-17 and IFN-γ and showed a memory phenotype. Locally enriched IL-22⁺CD4⁺ T cells positively correlated with increased CD14⁺ monocytes and IL-6 and IL-23 detection ex vivo, and in vitro IL-6 and IL-23 induced the polarization of IL-22⁺CD4⁺ T cells in a dose-dependent manner and the polarized IL-22⁺CD4⁺ T cells co-expressed of IL-17 and IFN-γ. Moreover, IL-22⁺CD4⁺ T-cell subsets (IL-22⁺IL-17⁺CD4⁺, IL-22⁺IL-17^?CD4⁺, IL-22⁺IFN-γ⁺CD4⁺, IL-22⁺IFN-γ^?CD4⁺, and IL-22⁺IL-17⁺IFN-γ⁺CD4⁺ T cells), and Th22 cells were also increased in tumors. Furthermore, higher intratumoral IL-22⁺CD4⁺ T-cell percentage and Th22-cell percentage were found in patients with tumor-node-metastasis stage advanced and predicted reduced overall survival. In conclusion, our data indicate that IL-22⁺CD4⁺ T cells and Th22 cells are likely important in establishing the tumor microenvironment for GC; increased intratumoral IL-22⁺CD4⁺ T cells and Th22 cells are associated with tumor progression and predict poorer patient survival, suggesting that tumor-infiltrating IL-22⁺CD4⁺ T cells and Th22 cells may be suitable therapeutic targets in patients with GC.     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ～30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.     

In vivo determination of mouse olfactory mucus cation concentrations in normal and inflammatory States

Objective

Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans.

Study Design

Animal study in mice and translational study in humans.

Methods

Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin’ Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations.

Results

In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na⁺] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K⁺] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca²⁺] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75).

Conclusion

Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.     

胸膜肺炎放线杆菌RTX毒素与宿主的相互作用

摘要：胸膜肺炎放线杆菌引起猪传染性胸膜肺炎,给养猪业造成严重的经济损失。RTX毒素是胸膜肺炎放线杆菌主要的毒力因子,在该病原的感染与免疫中发挥“双刃剑”的作用。本文综述了近十多年来国内外在胸膜肺炎放线杆菌RTX毒素的研究进展,提出了毒素与宿主互作研究的必要性和技术可行性,认为毒素与宿主相互作用研究将诠释此病原的分子致病机理。