Liraglutide Promotes the Osteogenic Differentiation in MC3T3-E1 Cells via Regulating the Expression of Smad2/3 Through PI3K/Akt and Wnt/ß-Catenin Pathways.

Diabetes is a worldwide health problem with increasing prevalence. Some reports indicate the interplay between bone and glucose metabolism. The imbalance between bone resorption and formation resulted in the structural integrity and strength of bone. Glucagon-like peptide-1 (GLP-1) and its agonists (Liraglutide) have an anabolic action on bone remodeling by stimulating osteoblast differentiation as well as increasing osteoblast longevity. However, the underlying mechanisms remain elusive. We detected the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells via immunocytochemistry assay. Alkaline phosphatase activity assay, alizarin red stain, quantitative real-time polymerase chain reaction, and western blot were employed to detect the effect of Liraglutide on osteogenic differentiation. Liraglutide promoted the expression of GLP-1R in a dosage- and time-dependent manner, and it enhanced the osteogenic differentiation in MC3T3-E1 cells. Liraglutide application improved the levels of Smad2/3 and p-Smad2/3; however, the silencing of Smad2/3 blocked the osteogenic differentiation induced by Liraglutide. What is more, the application of PI3K and Wnt inhibitors inhibited the upregulation of Akt, p-Akt, ß-catenin, Smad2/3, and p-Smad2/3 induced by Liraglutide. Liraglutide facilitated the osteogenic differentiation via the regulation of Smad2/3 via PI3K/AKT and Wnt/ß-catenin pathways. These data revealed a new mechanism of Liraglutide inducing osteogenic differentiation and provided theory evidence to maintain normal bone metabolism during diabetes therapy.

[Construction of quantitative real-time PCR detection system of transgenic tomato line Zeneca B,Da,F].

OBJECTIVE: To construct the plasmid reference molecules for detection of transgenic tomato line Zeneca B,Da,F using quantitative real-time PCR(qPCR). METHODS: Three plasmid reference molecules pEasy-T3-APX, pEasy-T3-16A and pEasy-T3-16S were cloned based on reverse genetics, which contain the target fragments of tomato endogenous reference gene apx (ERG-apx), gene-specific sequence of pg(GS-pg) and construct-specific sequence of vectors pJR16S/pJR16A (CS-16S/CS-16A) of Zeneca B,Da,F, respectively. Primers and Taqman probes were designed by Beacon Designer 7.5.The specificity, sensitivity, reproducibility and the limit of detection(LOD) of the qualitative and quantitative PCR system based on the plasmid reference molecules were evaluated. PicoGreen was used to measure the DNA concentration of the plasmid reference molecules. Two sets of samples containing 1% or 0.1% (w/w) pEasy-T3-16A or pEasy-T3-16S mixed with pEasy-T3- APX as background DNA were prepared for evaluating the efficacy of the qPCR system. RESULTS: The target fragments for qPCR detection were anchored, ERG-apx 108 bp, GS-pg 108 bp , CS-16S 109 bp and CS-16A 102 bp. The three plasmid reference molecules were confirmed at the expected sizes by restriction enzyme digestion. The qPCR results showed that the RSD of reproducibility were 0.2% to1.5%, LOD was 25 copies, R2 values for these standard curves were 0.994 ~0.998 and amplification efficiencies were 93.3%~102.4%.The bias between the test and true values of two sets of mixed samples ranged from -9.3% to 14.7% after adjusting by conversion factors(Cf). CONCLUSION: The plasmid reference molecules and qPCR system for qualitative and quantitative detection of transgenic tomato line Zeneca B,Da,F have been established successfully.

[Optimization of tomato genetic transformation, kanamycin-resistant screening and seed selection].

OBJECTIVE: To optimize the process of tomato genetic transformation, screening and seed selection using multiepitope antigenic gene (MAG) and truncated major surface antigen 1 (tSAG1) of Toxoplasma gondii as the target insert genes. METHODS: Tomato high-frequency regeneration system was optimized with different choices of media and explants. The genetic transformation procedure was optimized using different tomato cultivars, explants, culture temperatures, media and acetosyringone (AS) supplements. Three concentrations of kanamycin were utilized for resistant selection of the transgenic candidate roots. The selected lines were trained, transplanted to soil and grown in a greenhouse till maturity. Sterile seeding using kanamycin-incorporated medium was conducted for screening transgenic tomato generations. RESULTS AND CONCLUSION: Cotyledons were better than hypocotyls as the regeneration explants. The regeneration rate of cotyledons reached 98% (59/60) using the optimized regeneration medium ZB3. The culture medium and temperature were the key factors for tomato transgenic shoot induction. The number of transgenic buds increased significantly at the appropriate temperature condition (23-/+1 degrees celsius;), and AS of 100 micromol/L in the medium before inoculation also significantly raised transformation rate. The budding rate of Zhongshu No.5 cotyledons was 35% (28/81) using the medium ZB2 under (23-/+1) degrees celsius;. Kanamycin at 80 mg/L was optimal for transgenic plantlet rooting selection with the rooting rate of 48% (31/65). 117 transgenic lines were obtained. Non-transgenic tomato plant growth, especially the root and elongation, was inhibited obviously with kanamycin at 100 mg/L or above, and the roots became purple and lacked lateral roots. The transgenic tomato seeds could be selected effectively with kanamycin at 150 mg/L.

Automatic separation system for marine meiobenthos based on laser-induced fluorescence technology.

An automatic system for marine meiobenthos separation was developed by using laser-induced fluorescence technology. Rose Bengal was used as organism dye and the spectrums of Rose Bengal were measured. Laser-induced fluorescence system was established to detect marine meiobenthos in sediments. Data obtained from experiments were analyzed by using a mathematical model. The results showed that laser-induced fluorescence technology worked well in the system. The system could select the meiobenthos efficiently and precisely.

Microarray-based rapid cloning of an ion accumulation deletion mutant in Arabidopsis thaliana.

Here we describe the development of a microarray-based mapping strategy to rapidly isolate deletion mutant genes. The presented approach is particularly useful for mapping mutant genes that are difficult to phenotype. This strategy uses masking bulk segregant analysis to mask unrelated deletions, thus allowing identification of target deletions by microarray hybridization of pooled genomic DNA from both WT and mutant F2 populations. Elemental profiling has proven to be a powerful tool for isolation of nutrient and toxic metal accumulation mutants in Arabidopsis. Using microarray mapping, a sodium overaccumulation mutant FN1148 was identified as having a 523-bp genomic deletion within the second exon and intron of the AtHKT1 gene. Further cosegregation, complementation, and comparative analyses among different salt-sensitive mutants confirmed that the deletion within the AtHKT1 gene is responsible for the sodium overaccumulation in shoots and leaf sodium sensitivity of the FN1148 mutant. These results demonstrate that microarray-based cloning is an efficient and powerful tool to rapidly clone ion accumulation or other genetic deletion mutants that are otherwise difficult to phenotype for mapping, such as metabolic or cell signaling mutants.