Multiwalled carbon nanotubes co-delivering sorafenib and epidermal growth factor receptor siRNA enhanced tumor-suppressing effect on liver cancer.

OBJECTIVE: This study aimed to investigate the effects of multiwalled carbon nanotubes (MWNTs) co-delivering sorafenib (Sor) and epidermal growth factor receptor (EGFR) siRNA (MWNT/Sor/siRNA) on tumor growth in liver cancer (LC). RESULTS: MWNT/Sor/siRNA was proved to possess increased Sor release, high siRNA stability, and enhanced cellular uptake. In addition, MWNT treatment has few effects on cell proliferation and apoptosis in HepG2 cells; however, MWNT/Sor/siRNA treatment significantly inhibited clone number and induced cell apoptosis, which shows a more favorable antitumor effect than MWNT/Sor and free Sor and free siRNA in HepG2 cells. Moreover MWNT/Sor/siRNA treatment has the most significant antitumor effect in vivo. CONCLUSIONS: MWNT/Sor/siRNA exhibited a superior antitumor effect in vitro and in vivo. METHODS: The MWNT/Sor and MWNT/Sor/siRNA were prepared, and then the morphologies of MWNT/Sor/siRNA were analyzed. In vitro Sor release assay, siRNA stability and cellular uptake of MWNT/Sor/siRNA were performed as well. Next, the effects of MWNT, free Sor, free siRNA, MWNT/Sor and MWNT/Sor/siRNA were evaluated by colony-forming assay, and cell apoptosis assay in HepG2 cells. Meanwhile, the level of EGFR and proteins associated with apoptosis was tested. Furthermore, the anti-tumor effects of MWNT/Sor/siRNA on LC xenograft mice were also unraveled.

Integrative Analysis of the Wheat PHT1 Gene Family Reveals A Novel Member Involved in Arbuscular Mycorrhizal Phosphate Transport and Immunity.

Phosphorus (P) deficiency is one of the main growth-limiting factors for plants. However, arbuscular mycorrhizal (AM) symbiosis can significantly promote P uptake. Generally, PHT1 transporters play key roles in plants' P uptake, and thus, PHT1 genes have been investigated in some plants, but the regulation and functions of these genes in wheat (TaPHT1) during AM symbiosis have not been studied in depth. Therefore, a comprehensive analysis of TaPHT1 genes was performed, including sequence, phylogeny, cis-elements, expression, subcellular localization and functions, to elucidate their roles in AM-associated phosphate transport and immunity. In total, 35 TaPHT1s were identified in the latest high-quality bread wheat genome, 34 of which were unevenly distributed on 13 chromosomes, and divided into five groups. Sequence analysis indicated that there are 11 types of motif architectures and five types of exon-intron structures in the TaPHT1 family. Duplication mode analysis indicated that the TaPHT1 family has expanded mainly through segmental and tandem duplication events, and that all duplicated gene pairs have been under purifying selection. Transcription analysis of the 35 TaPHT1s revealed that not only known the mycorrhizal-specific genes TaPht-myc, TaPT15-4B (TaPT11) and TaPT19-4D (TaPT10), but also four novel mycorrhizal-specific/inducible genes (TaPT3-2D, TaPT11-4A, TaPT29-6A, and TaPT31-7A) are highly up-regulated in AM wheat roots. Furthermore, the mycorrhizal-specific/inducible genes are significantly induced in wheat roots at different stages of infection by colonizing fungi. Transient Agrobacterium tumefaciens-mediated transformation expression in onion epidermal cells showed that TaPT29-6A is a membrane-localized protein. In contrast to other AM-specific/inducible PHT1 genes, TaPT29-6A is apparently required for the symbiotic and direct Pi pathway. TaPT29-6A-silenced lines exhibited reduced levels of AM fungal colonization and arbuscules, but increased susceptibility to biotrophic, hemi-biotrophic and necrotrophic pathogens. In conclusion, TaPT29-6A was not only essential for the AM symbiosis, but also played vital roles in immunity.

Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses.

Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells.

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a ß-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.