Preserving Porcine Genetics: A Simple and Effective Method for On-Site Cryopreservation of Ear Tissue Using Direct Cover Vitrification.

Cell cryopreservation is widely used for porcine genetic conservation; however, isolating and freezing primary cells in farms without adequate experimental equipment and environment poses a significant challenge. Therefore, it is necessary to establish a quick and simple method to freeze tissues on-site, which can be used for deriving primary fibroblasts when needed to achieve porcine genetic conservation. In this study, we explored a suitable approach for porcine ear tissue cryopreservation. The porcine ear tissues were cut into strips and frozen by direct cover vitrification (DCV) in the cryoprotectant solution with 15% EG, 15% DMSO and 0.1 M trehalose. Histological analysis and ultrastructural evaluation revealed that thawed tissues had normal tissue structure. More importantly, viable fibroblasts could be derived from these tissues frozen in liquid nitrogen for up to 6 months. Cells derived from thawed tissues did not show any cell apoptosis, had normal karyotypes and could be used for nuclear transfer. These results suggest that this quick and simple ear tissue cryopreservation method can be applied for porcine genetic conservation, especially in the face of a deadly emerging disease in pigs.

Cytotoxic Nitrobenzoyloxy-substituted Sesquiterpenes from Spongederived Endozoic Fungus Aspergillus insulicola MD10-2.

The emergence of drug resistance and spread of new infectious diseases necessitated the development of novel antibiotics. Marine sponge-associated fungi represent a reservoir of novel molecules with diverse biological potentials. In this study, we isolated five nitrobenzoyloxy-substituted sesquiterpenes 1-5 from the culture mycelia of an endozoic fungus Aspergillus insulicola MD10-2, obtained from the South China Sea sponge Cinachyrella australiensis. Compound 2 showed cytotoxicity against human lung cancer cell line H-460 with an IC50 value of 6.9 µM. Cytotoxicity of the acetylated derivatives (2a and 2b) of compound 2 decreased markedly, suggesting that the hydroxyl group contributed to the cytotoxic activity. Compound 5 was inactive against H-460, which implied the double bond at C-7 had an effect on cytotoxic activity as well.

[Optimization of two-dimensional electrophoresis conditions for proteomics of Issatchenkia orientalis in degrading dyes].

Total protein of the yeast Issatchenkia orientalis was extracted and separated by two-dimensional electrophoresis (2-DE) before and after the dye Reactive Brilliant Red K-2BP was degraded by this yeast, respectively. Different protein extraction methods, different volumes of sample loaded and different staining techniques were tested and compared for the 2-DE. Among three different protein extraction methods, the final protein concentrations of 3.4 mg/mL, 1.8 mg/mL and 5.6 mg/mL were obtained by single ultrasonication, ultrasonication-TCA/acetone,and ultrasonication-ammonium sulfate precipitation, respectively. The best electrophoresis pattern could be gotten by loading 150 microg protein samples from the method of ultrasonication-ammonium sulfate precipitation, using IPG strips of pH 4-7 for the first dimensional electrophoresis and staining with silver nitrate. This electrophoresis pattern had high resolution and good repetition. It was detected to have 730 +/- 30 protein points by preliminary image analysis. This research results provided a technical support for screening dye-degrading enzymes from the yeast of I. orientalis.

[Decolorization of synthetic dyes with a new isolated yeast Candida krusei].

A new yeast isolate Y-G-1, identified as Candida krusei, capable of degrading Reactive Brilliant Red K-2BP, was obtained from soil by screening experiments. This strain gave a maximal decolorization (99%) for Reactive Brilliant Red K-2BP (200 mg/L) after incubation of 12 h. In order to get this decolorization effect, the optimal inoculation volume of Candida krusei was not less than 5%, the optimal pH of culture medium was within a range from 4 to 9, and the concentrations of (NH4)2SO4 and glucose were not lower than 0.1% and 0.5%, respectively. The research results on the mechanism of decolorization show that Candida krusei could biodegrade Reactive Brilliant Red K-2BP. In addition, Candida krusei could also decolorize 62% - 96% of another 9 dyes (50mg/ L). Specially for azo dyes, including Weak Acid Brilliant Red B, Reactive Black KN-B and Reactive Red M-3BE, their decolorization rates were over 90 %, and for triphenylmethane dye, Mordant Blue B, it was up to 93%. All these results indicate that this strain have potential application in dye wastewater treatment.