When development is constantly but weakly perturbed - Canalization by microRNAs

Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of GRN (gene regulatory network) stabilization as well as developmental canalization against constant but small environmental perturbations. Since the complete removal of miRNAs is lethal, we construct a Dicer-1 knockdown line (dcr-1 KD) in Drosophila that modestly reduces all miRNAs. We hypothesize that flies with modest miRNA reductions will gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in the dcr-1 KD line but, importantly, adult longevity is reduced by ∼ 90%. When flies are stressed by high temperature, dcr-1 KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. We further show that the developmental failure is associated with GRN aberration in the larval stages even before phenotypic aberrations become observable. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be the genome’s solution to weak but constant environmental perturbations.

Optimization of In Vitro Culture Conditions of Sturgeon Germ Cells for Purpose of Surrogate Production

To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz’s L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.

Production of Schisandrin A and Schisandrin B from Callus and Suspension Cell Cultures of Schisandra chinensis

For establishing the fermentation system of synthetic schizandrin A and schizandrin B, culture conditions of the Schisandra chinensis callus and the suspension cell were studied in this paper. The friable calluses of Schisandra chinensis (Turcz.) Baill. were induced from hypocotyls in Murashige-Skoog (MS) solidified medium supplemented with hormone 6-benzylaminopurine (6-BA) and 2,4-Dichlorophenoxyacetic butyl acetate (2,4-D) in different concentrations, and suspension cells initiated from friable callus were cultured in MS liquid medium with various concentrations and combinations of 6-BA, 2,4-D and kinetin (KT). The optimal culture condition for callus inducement was found to be MS solidified medium with 6-BA 1.0 mg/l and 2,4-D 0.3 mg/l and the optimal condition for suspension cells was MS liquid medium with 6-BA 1.0 mg/l, 2,4-D 0.2 mg/l and KT 0.5 mg/l. UPLC/Q-TOF-MS method was used for accurate identification of schisandrin A and schisandrin B in the seeds, callus and suspension cells of S. chinensis. And HPLC analytical method was used successfully for identification and quantification of these metabolites in cultures of S. chinensis. As a result, schisandrin A was obtained 0.251 mg/g, 0.118 mg/g and 0.115 mg/g from seeds, callus and suspension cells and schisandrin B was 0.142 mg/g, 0.086 mg/g and 0.05 mg/g from seeds, callus and suspension cells, respectively. Our datas indicate that callus and suspension cells had capability as seeds of S. chinensis for schisandrin A and schisandrin B synthesizing and can be used as potential sources of these biologically active lignans.

Production of a Novel Biopolymer by Culture of Pseudomonas fluorescens Using Brewery Wastewater and its Use for Dye Removal

The optimal conditions of production of biopolymer by the culture of Pseudomonas fluorescens were examined, using brewery wastewater to replace glucose as carbon source and energy source in the culture medium. Results showed that the COD concentration in brewery wastewater favorable for the production of the biopolymer was 6000 mg•L-1, and an optimal culture condition of inoculum size of 5%(v/v), 28°C, initial pH 7.0 and shaking speed of 150 r•min-1, under the optimal culture conditions, the highest flocculating activity achieved for Kaolin suspension was 97.5% and 3.67 g biopolymer /L broth was obtained. The biopolymer was effective in flocculating some soluble reactive dyes in aqueous solution, reactive Light-Yellow K-4G and reactive Turquoise Blue KN-G with a decolorization efficiency of 95.8 and 96.2%, respectively，using 25 mL of the flocculant in 500 mL of 100 mg•L-1 dye solution.The decolorization efficiency was dependent on the flocculant dosage and solution pH.