Study of Sexual-Linked Genes (OGI and MeGI) on the Performance of Androecious Persimmons (Diospyros kaki Thunb.)

It is reported that the production of floral sexual phenotype in hexaploid monoecious persimmon (Diospyros kaki) is closely related to a pseudogene called OGI, and a short interspersed nuclear element (SINE)-like insertion (named Kali) in the OGI promoter leads to the gene silence. As a result, DNA methylation level of MeGI promoter determines the development of male or female flowers. However, the molecular mechanism in androecious D. kaki, which only bear male flowers, remains elusive. Here, real-time quantitative polymerase chain reaction (RT-qPCR), molecular cloning, and bisulfite PCR sequencing technique were carried out using 87 materials, including 56 androecious resources, 15 monoecious, and 16 gynoecious cultivars, to investigate the performance of OGI and MeGI on the specific androecious type of D. kaki in China. In conclusion, the Kali insertion was exactly located in the OGI promoter region, and the OGI gene and the Kali sequence were existing and conserved in androecious D. kaki. Meanwhile, we also demonstrated that the MeGI gene was widespread in our investigated samples. Ultimately, our result convincingly provided evidence that the low expression of OGI is probably ascribed to the presence of Kali displaying strong methylation in the OGI promoter, and low expression of MeGI, as well as high DNA methylation level, in the promoter was closely connected with the production of male flowers; this result was consistent with the monoecious persimmon model. Our findings provide predominant genetic aspects for investigation into androecious D. kaki, and future perfecting the sex-determining mechanisms in persimmon.

A MarR-family transcriptional factor MapR positively regulates actinorhodin production in Streptomyces coelicolor

ABSTRACT Production of secondary metabolites is tightly regulated by transcriptional regulators in Streptomyces. Many regulators have been identified by gene disruption in Streptomyces; however, there are still unknown regulatory genes yet to be revealed due to gene silence under laboratory culture conditions. In present study, a T7 expression system was used to identify novel regulators by gene deletion and constitutive-expression of nine candidates. We reported that the constitutive-expression of a MarR family transcriptional regulator MapR (SCO2398) resulted in increase of actinorhodin (ACT) production. The expression profiling indicated that MapR regulates ACT production through a transcriptional cascade. Collectively, our study suggests that MapR serves as a novel positive regulator of ACT production.

Inhibition of PI3K/AKT molecular pathway mediated by membrane estrogen receptor GPER accounts for cryptotanshinone induced antiproliferative effect on breast cancer SKBR-3 cells

Background : Breast cancer is the most frequently diagnosed cancer among women and the second leading cause of cancer death. Especially nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. In recent years, it was reported that membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor took essential part in the development and treatment of breast cancer, especially nER negative breast cancer. The present study aimed to investigate the anti nER negative breast cancer effect and its possible molecular pathway of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen.Methods: The following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT. Results: The results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT also exerted antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G1 and attenuated by G15. Results of western blot and immunofluorescence both showed that PI3K and p-AKT protein expression could be down regulated by CPT and such effects were mediated by GPER which is further demonstrated by GPER gene silence test.Conclusion: Our present study shows that the antiproliferative action of CPT on SKBR-3 cells is realized by inhibition of GPER mediated PI3K/AKT pathway. These findings deserve further validation for serving as useful therapeutic targets.

Enhancing PD-1 Gene Silence in T Lymphocytes by Comparing the Delivery Performance of Two Inorganic Nanoparticle Platforms

Suitable carriers are crucial to RNAi applications for cancer genotherapy and T-cell immunotherapy. In this research, we selected two extensively-investigated biocompatible inorganic nanoparticle carriers, i.e., layered double hydroxide (LDH) and lipid-coated calcium phosphate (LCP) and then compared their efficacy for siRNA delivery in T cells, in order to understand which carrier is more efficient in delivering functional programmed cell death protein 1 siRNA (PD-1 siRNA) to suspended T lymphocytes. Both LDH and LCP nanoparticles quickly delivered gene segment to mouse T cell lines (EL4), while the LCP nanoparticles exhibited more cellular uptake and higher PD-1 gene silence efficiency. We further demonstrated that LCP nanoparticles successfully reduced the expression of PD-1 in human ex vivo tumor infiltrating lymphocytes (TILs). Thus, LCP nanoparticles can be used as a better nano-carrier for gene therapy in lymphocytes, especially in regards to TIL-related cancer immunotherapy.

Abstract 16665: Caveolin-1 and α7nachr Interaction is Essential in Preventing Nicotine Induced Vascular Inflammatory Response in Mice

Nicotine is an independent risk factor of and associated with the development of atherosclerosis. Dysfunction of endothelial Cavolin-1 (Cav-1) can accelerate atherosclerosis progression via boosting inflammation response. However, the causative link between nicotine and atherosclerosis remains unknown. Here, we utilized molecular and cellular experiments revealed that in human endothelial cells (HUVECs), 10-6 M nicotine increased intracellular oxygen free radical generation, inhibited eNOS activity, augmented pathological NO production from iNOS, promoted nitrative stress (Nitrotyrosine), and increased adhesion molecule expression when compared with vehicle. In vivo administration of nicotine upregulated plasma adhesion molecule levels and significantly increased nitrotyrosine levels in vascular tissue (2±0.4 fold compared with untreated group, p<0.05). Bioinformatics analysis predicted that α7nAChR motif (151YIDVRWFPF159 and 168FGSWSYGGW176) may interact with Cav-1 via Cav-1 scaffolding domain (82DGIWKASFTTFTVTKYWFYR101). The bioinformatic analysis was confirmed by immunocytochemistry and immunoprecipitation showing that α7nAChR co-localizes with caveolin-1 (Cav-1) and these two molecules interact reciprocally. Although 10-6M nicotine did not alter expression levels of Cav-1, it significantly decreased the interaction between Cav-1 and nicotine receptor (α7nAchR). After siRNA Cav-1 gene silence, nicotine treatment further significantly increased ROS generation (1.37 + 0.33 times vs. scrambled siRNA, p<0.05), enhanced pathological NO production (1.35 ± 0.4 fold, p<0.05), and increased adhesion molecule expression (1.4 ±0.4 fold, p<0.05). AMPKα1, a sensor of oxidative stress, was phosphorylated after nicotine treatment. However, siRNA AMPK gene silence failed to block nicotine induced adhesion molecule expression. Taken together, these results demonstrated that reduced α7nAchR/Cav-1interaction plays an important role in nicotine induced endothelial inflammatory response; this inflammation signal pathway is AMPK independent. Preserving and strengthening stability of Cav- 1/α7nAchR interaction is a potential target in treatment of vascular dysfunction caused by smoking.