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1.  Isolation and Characterization of a Novel Endoglucanase from a Bursaphelenchus xylophilus Metagenomic Library 
PLoS ONE  2013;8(12):e82437.
A novel gene (designated as cen219) encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50°C and 6.0. It was stable from 30 to 50°C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by detergent SDS and metals Fe3+, Cu2+ or Hg2+. The enzyme hydrolyzed a wide range of β-1, 3-, and β-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The Km and Vmax of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host–parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.
PMCID: PMC3873927  PMID: 24386096
2.  The ComP-ComA Quorum System Is Essential For “Trojan horse” Like Pathogenesis in Bacillus nematocida 
PLoS ONE  2013;8(10):e76920.
Bacillus nematocida B16 has been shown to use “Trojan horse” mechanism in pathogenesis that has characteristics of “social” behavior. The ComP-ComA system, a conserved quorum sensing system in the genus Bacillus, functions in many physiological processes including competence development, lipopeptide antibiotic surfactin production, degradative enzyme production and even some unknown functions. Here we investigated the requirement of ComP-ComA system in B. nematocida B16 for its pathogenicity against nematodes. The ΔcomP mutant displayed deficiencies in attracting and killing nematodes, due to the absence of attractive signal molecules and the decreased expressions of virulence factors, respectively. Contrarily, a complemented comP mutant at least partially resumed its pathogenicity. Our data from transcriptional analysis further confirmed that this signaling system directly or indirectly regulated the expressions of two major virulence proteases in the infection of B. nematocida B16. Bioinformatics analyses from comparative genomics also suggested that the potential target genes of transcription factor ComA were involved in the processes such as the synthesis of attractants, production of extracellular degradative enzymes and sortase, secondary metabolites biosynthesis, regulation of transcription factors, mobility, as well as transporters, most of which were different from a saprophytic relative B. subtilis 168. Therefore, our investigation firstly revealed that the participation and necessity of ComP-ComA signaling system in bacterial pathogenesis.
PMCID: PMC3793909  PMID: 24130811
3.  Pathway and Molecular Mechanisms for Malachite Green Biodegradation in Exiguobacterium sp. MG2 
PLoS ONE  2012;7(12):e51808.
Malachite green (MG), N-methylated diaminotriphenylmethane, is one of the most common dyes in textile industry and has also been used as an effective antifungal agent. However, due to its negative impact on the environment and carcinogenic effects to mammalian cells, there is a significant interest in developing microbial agents to degrade this type of recalcitrant molecules. Here, an Exiguobacterium sp. MG2 was isolated from a river in Yunnan Province of China as one of the best malachite green degraders. This strain had a high decolorization capability even at the concentration of 2500 mg/l and maintained its stable activity within the pH range from 5.0 to 9.0. High-pressure liquid chromatography, liquid chromatography-mass spectrometry and gas chromatography–mass spectrometry were employed to detect the catabolic pathway of MG. Six intermediate products were identified and a potential biodegradation pathway was proposed. This pathway involves a series of reactions of N-demethylation, reduction, benzene ring-removal, and oxidation, which eventually converted N-methylated diaminotriphenylmethane into N, N-dimethylaniline that is the key precursor to MG. Furthermore, our molecular biology experiments suggested that both triphenylmethane reductase gene tmr and cytochrome P450 participated in MG degradation, consistent with their roles in the proposed pathway. Collectively, our investigation is the first report on a biodegradation pathway of triphenylmethane dye MG in bacteria.
PMCID: PMC3522578  PMID: 23251629
4.  Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation 
PLoS Pathogens  2011;7(9):e1002179.
Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.
Author Summary
The fungus Arthrobotrys oligospora has multiple lifestyles. It's not only a nematode pathogen, but also a saprophyte, a pathogen of other fungi, and a colonizer of plant roots. As a nematode pathogen, A. oligospora forms adhesive networks to capture nematodes and is a model organism for understanding the interaction between these fungi and their host nematodes. In this study, the whole genome sequence of A. oligospora was reported. Our analyses of the proteome profiles of intracellular proteins from cells treated with nematode extracts for 10 h and 48 h revealed a key set of genes involved in trap formation. The changes in protein levels for some trap formation related genes were further confirmed by qPCR. The combined genome and proteome analysis identified the major genetic and metabolic pathways involved in trap formation in A. oligospora. Our results provide the first glimpse into the genome and proteome of this fascinating group of carnivorous fungi. The data should serve as a roadmap for further investigations into the interaction between nematode-trapping fungi and their host nematodes, providing broad foundations for research on the biocontrol of pathogenic nematodes.
PMCID: PMC3164635  PMID: 21909256
5.  Clinicopathological Characteristics and Her-2/neu Status in Chinese Patients with Uterine Papillary Serous Carcinoma 
ISRN Obstetrics and Gynecology  2010;2011:575327.
Objective. To analyze clinico-pathological features of Chinese patients with UPSC, and investigate roles of Her-2/neu protein expression and gene amplification in UPSC prognosis. Methods. Thirty-six patients with UPSC treated in Cancer Hospital of Fudan University from 1996 to 2006 were analysed retrospectively. Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) were performed to evaluate Her-2/neu gene amplification and protein expression respectively. Results. The median age was 63 years, and 61% (22/36) were late stages (stage III/IV). The 1-year, 3-year, and 5-year overall survival (OS) was 73.1%, 51.9% and 43.9%, respectively. Advanced stages (P = .0006) and deep myometrial invasion (P = .0138) were significantly associtated with a shorter OS. In 36 cases, 27.8% (10/36) showed 2+ staining and 8.3% (3/36) showed 3+ by IHC. Amplification of the Her-2/neu gene was observed in 11.1% (4/36) cases. The 5-year overall survival rate in Her-2/neu IHC 2 + ∼3+ and 0 ~ 1+ cases was 12.9% and 68.6% respectively. Her-2/neu protein expression 2 + ∼3+ was significantly associated with advanced surgical stage and worse overall survival (P = .03 and P = .0023, resp.). Conclusion. Chinese patients with UPSC showed characteristics of deep myometrial invasion, advanced stages and poor overall survival. Her-2/neu protein overexpression is associated with advanced stage and poor survival outcome.
PMCID: PMC3102014  PMID: 21647234

Results 1-5 (5)