Effect of siRNA targeting EZH2 on cell viability and apoptosis of bladder cancer T24 cells.

We investigated the effect of siRNA targeting enhancer of EZH2 on cell proliferation, invasion, migration, and apoptosis of human bladder cancer T24 cells. An siRNA-expressing plasmid targeting the EZH2 gene was transfected into T24 cells. Quantitative polymerase chain reaction and Western blot analysis were used to detect EZH2 expression at the mRNA and protein levels, respectively. Proliferation, invasion, and migration of T24 cells were examined in vivo using MTT, wound healing, and transwell chamber migration assays, respectively. Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometric analysis was performed to determine cell apoptosis levels. Expression of EZH2 in T24 cells was suppressed at the mRNA and protein levels. Following transfection for 48 h, growth was inhibited by 37.9%, which was markedly lower than that in the negative control group (P < 0.05). Following a wound-healing assay for 24 h, transfected cell migration distance was 1.37 ± 0.12, which was markedly less than the horizontal migration distance of negative control group cells (P < 0.01). In addition, the cell invasion ability of EZH2- siRNA group cells decreased by 67% compared with negative control group cells (P < 0.01). Following transfection for 48 h, early- and late-stage apoptosis rates for T24 cells were 22.8 and 3.60%, respectively, which were higher than in the negative control group (P < 0.01). EZH2 gene silencing effectively suppressed the proliferation, invasion, and migration abilities of human bladder cancer cells, promoting apoptosis.

First Report of a Leaf Spot on Goldthread (Coptis chinensis) Caused by Phoma aquilegiicola in China.

Goldthread (Coptis chinensis) is an important herbaceous plant in traditional Chinese medicine (3). Annual production of goldthread root is ~3,000 tons (dry weight) in China. The plant is cultivated extensively in Shizhu Co., Chongqing (29.98°E, 108.13°N), where goldthread yields account for more than 60% of total world production. A foliar disease was first observed on goldthread plants in 2008 in Shizhu County (5). In 2011 and 2012, about 10 ha of goldthread fields in different townships of Shizhu Co. were surveyed. The results demonstrated that the disease present in the fields was widespread at incidences of 30 to 100%, with yield losses of 15 to 75%. Typical symptoms included irregular, purple brown lesions on leaves, beginning usually at the leaf margin and extending to the central leaf blade. The lesions coalesced and turned deep purple. Black pycnidia were visible on the lesions, and severely diseased plants were usually wholly blighted. To identify the pathogen, infected leaves were collected from goldthread fields in different townships of Shizhu Co. and small pieces of symptomatic tissue were cut from each leaf. The leaf pieces were surface-disinfected for 1 min in 1.5% sodium hypochlorite, rinsed in sterilized water, air-dried, and transferred onto potato dextrose agar (PDA) plates with 0.5 g/liter of streptomycin sulfate. Thirty-three fungal isolates with similar colony morphology were obtained. On oatmeal agar plates, each colony was circular with a smooth edge, initially cream, and then pale-brown. Pycnidia were dark brown, spherical, with or without papillae, and 100 to 112 × 189 to 222 µm. Conidia were produced on short, straight, and aseptate conidiophores in the pycnidia; they were monocellular, hyaline, ellipsoidal or clavate, and 2.01 to 2.50 × 4.20 to 5.55 µm. Three isolates (SZ-9, SZ-10, and SZ-13) were selected randomly from all 33 isolates, and genomic DNA of each isolate was extracted following the CTAB method (4). The rDNA ITS region of each isolate was amplified with V9G/ITS4 primers and sequenced (1). The ITS sequences of the three isolates (GenBank Accession Nos. KF692355.2 [SZ-9], KF985236.1 [SZ-10], and KF985237.1 [SZ-13]) were identical, and BLAST revealed 100% identity with the ITS sequence of an isolate of Phoma aquilegiicola (CBS 107.96, GU237735.1). Based on the morphological characteristics and ITS sequences, all three isolates were identified as P. aquilegiicola. Pathogenicity test of 10 isolates was conducted by placing a 5-mm-diameter mycelial agar plug (from the margin of a 5-day-old PDA culture) on each of 10 fully-expanded leaves of healthy goldthread plants/isolate. Ten leaves were treated similarly with sterilized PDA plugs as a control. Inoculated and control plants were incubated in the dark for 24 h at 25 ± 2°C and >90% RH, and then maintained in a growth chamber at 25 ± 2°C, 3,100 lux, and >90% RH. The pathogenicity test was carried out three times. Symptoms developed on all inoculated leaves for all 10 isolates, but not on the control plants. Lesions were first visible 48 h after inoculation, and typical irregular lesions similar to those observed on field plants were seen after 6 days. The same pathogenic fungus was re-isolated from the infected leaves but not from the non-inoculated leaves. A disease caused by P. aquilegiicola was first reported on Aquilegia flabellata plants of the cv. Fan Columbine in a perennial garden in Italy (2). This is the first report of leaf spot on goldthread caused by P. aquilegiicola in China. Studies on the epidemiology and control of the disease are necessary owing to the economic significance of the host and destructiveness of the disease. References: (1) M. M. Aveskamp et al. Mycologia 101:363, 2009. (2) A. Garibaldi et al. Plant Dis. 95:880, 2011. (3) B. Liu et al. J. Pharmaceut. Biomed. 41:1056, 2006. (4) M. A. Saghai-Maaroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984. (5) X. R. Zhou et al. J. Shizhen Medicine Res. 23:471, 2012.

Genetic diversity among red swamp crayfish (Procambarus clarkii) populations in the middle and lower reaches of the Yangtze River based on AFLP markers.

The red swamp crayfish has become one of the most important freshwater aquaculture species in China. At present, although it is widely distributed in the middle and lower reaches of the Yangze River basin, little is known about its population genetics and geographic distribution in China. We estimated the genetic diversity among 6 crayfish populations from 4 lakes (Hongze Lake, Poyang Lake, Dongting Lake, and Yue Lake) using AFLPs. A total of 129 loci were generated with 5 EcoRI-MseI primer combinations and scored as binary data in 139 individuals. These data were analyzed by cluster methods with the NTSYSpc software package. The 6 populations were separated into 3 major clusters by principal coordinate analysis and cluster analysis. Among the 6 populations, the highest gene diversity was found within the Nanjing population. Analysis of molecular variance demonstrated that most variation occurred within populations (91.20%). The estimated average GST value across all loci was 0.4186, suggesting (very) low gene flow among the different localities. We conclude that there is high genetic differentiation among crayfish in the middle and lower reaches of the Yangze River. This information will help in the selection of high-quality individuals for artificial reproduction.