Computationally Reconstructed Interactome of Bradyrhizobium diazoefficiens USDA110 Reveals Novel Functional Modules and Protein Hubs for Symbiotic Nitrogen Fixation.

Symbiotic nitrogen fixation is an important part of the nitrogen biogeochemical cycles and the main nitrogen source of the biosphere. As a classical model system for symbiotic nitrogen fixation, rhizobium-legume systems have been studied elaborately for decades. Details about the molecular mechanisms of the communication and coordination between rhizobia and host plants is becoming clearer. For more systematic insights, there is an increasing demand for new studies integrating multiomics information. Here, we present a comprehensive computational framework integrating the reconstructed protein interactome of B. diazoefficiens USDA110 with its transcriptome and proteome data to study the complex protein-protein interaction (PPI) network involved in the symbiosis system. We reconstructed the interactome of B. diazoefficiens USDA110 by computational approaches. Based on the comparison of interactomes between B. diazoefficiens USDA110 and other rhizobia, we inferred that the slow growth of B. diazoefficiens USDA110 may be due to the requirement of more protein modifications, and we further identified 36 conserved functional PPI modules. Integrated with transcriptome and proteome data, interactomes representing free-living cell and symbiotic nitrogen-fixing (SNF) bacteroid were obtained. Based on the SNF interactome, a core-sub-PPI-network for symbiotic nitrogen fixation was determined and nine novel functional modules and eleven key protein hubs playing key roles in symbiosis were identified. The reconstructed interactome of B. diazoefficiens USDA110 may serve as a valuable reference for studying the mechanism underlying the SNF system of rhizobia and legumes.

Combination of levetiracetam and IFN-α increased temozolomide efficacy in MGMT-positive glioma.

PURPOSE: Glioma, especially glioblastoma (GBM), is the most aggressive malignant brain tumor and its standard therapy is often ineffective because of temozolomide (TMZ) resistance. Reversal of the TMZ resistance might improve the prognosis of glioma patients. We previously found that interferon-α (IFN-α) and anti-epileptic drug levetiracetam (LEV) could sensitize glioma to TMZ, respectively. In this study, we further investigated the efficiency of combining of LEV and IFN-α for improving the efficacy of TMZ. METHODS: We evaluated whether LEV and IFN-α could increase TMZ efficacy using colony formation assay and cell viability assay with MGMT-positive and MGMT-negative glioma cell lines in vitro. Subcutaneous xenografts and orthotopic xenografts mice models were used in vivo to observe the tumor growth and mice survival upon treatments with TMZ, TMZ + IFN-α, TMZ + LEV, or TMZ + LEV + IFN-α. The expression levels of MGMT, markers of pro-apoptotic and anti-apoptotic in tumor samples were analyzed by Western blotting. RESULTS: The combinational use of IFN-α, LEV, and TMZ showed the best anti-tumor activity in MGMT-positive cell lines (U138, GSC-1, U118, and T98 G). TMZ + LEV + IFN-α further obviously increased TMZ + LEV or TMZ + IFN-α efficiency in MGMT-positive cell lines, while not in negative cell lines (SKMG-4, U87, U373, and U251) in vitro, which were also observed in subcutaneous mice models (U138, GSC-1 compared to SKMG-4, U87) and orthotopic models (GSC-1) in vivo. Strikingly, the combination of LEV and IFN-α together with TMZ significantly prolonged the survival of mice with orthotopic GSC-1 glioma. Furthermore, we confirmed that the combination of LEV and IFN-α enhanced the inhibition of MGMT and the activation of apoptosis in U138 tumor on the basis of TMZ treatment. CONCLUSIONS: The combination use of LEV and IFN-α could be an optimal method to overcome TMZ resistance through obvious MGMT inhibition in MGMT-positive glioma.

Cloning of neuromedin B and its receptor in the rabbit and generating a polyclonal antibody to the neuromedin B protein.

Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction.