Rapamycin and trametinib: a rational combination for treatment of NSCLC

Mammalian target of rapamycin (mTOR) is one of the most commonly activated pathways in human cancers, including lung cancer. Targeting mTOR with molecule inhibitors is considered as a useful therapeutic strategy. However, the results obtained from the clinical trials with the inhibitors so far have not met the original expectations, largely because of the drug resistance. Thus, combined or multiple drug therapy can bring about more favorable clinical outcomes. Here, we found that activation of ERK pathway was responsible for rapamycin drug resistance in non-small-cell lung cancer (NSCLC) cells. Accordingly, rapamycin-resistant NSCLC cells were more sensitive to ERK inhibitor (ERKi), trametinib, and in turn, trametinib-resistant NSCLC cells were also susceptible to rapamycin. Combining rapamycin with trametinib led to a potent synergistic antitumor efficacy, which induced G1-phase cycle arrest and apoptosis. In addition, rapamycin synergized with another ERKi, MEK162, and in turn, trametinib synergized with other mTORi, Torin1 and OSI-027. Mechanistically, rapamycin in combination with trametinib resulted in a greater decrease of phosphorylation of AKT, ERK, mTOR and 4EBP1. In xenograft mouse model, co-administration of rapamycin and trametinib caused a substantial suppression in tumor growth without obvious drug toxicity. Overall, our study identifies a reasonable combined strategy for treatment of NSCLC.     

Developmental characteristics of floral organs and pollen of Chinese cabbage (Brassica campestris L. ssp. chinensis)

Morphological and cytological studies are complementary approaches to understand the molecular mechanisms that regulate floral developmental pathways. To better understand abnormal mutant phenotypes in floral development, we conducted detailed observations and investigations of the morphology, cytology, and cell ultrastructure of wild-type Chinese cabbage (Brassica campestris L. ssp. chinensis Makino and syn. B. rapa ssp. chinensis) flowers when they developed from primordia to anthesis. First, we measured bud and organ length with a stereo microscope and observed the developmental status and characteristics of the floral organs using a scanning electron microscope; then we made thin slices of anthers to observe the developmental stage and characteristics of pollen using an optical microscope; and finally, we made super-thin slices of anthers to observe the ultrastructure of pollen during its development with the aid of a transmission electron microscope. In this study, the floral developmental continuum was divided into 17 stages based on significant changes in the shape of floral primordia, and the pollen developmental continuum was divided into 14 stages based on the developmental characteristics. The results could provide the morphological basis for further research on the molecular mechanisms that regulate development of the floral organs and/or pollen of Chinese cabbage and their allied species.     

Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China

A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides.     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.     

Neospora caninum: Cloning and expression of a gene coding for cytokine-inducing profilin

Profilins are actin-binding proteins that in Toxoplasma gondii stimulate innate immunity in mice by binding Toll-like receptors (TLR) on dendritic cells (DC) leading to release of inflammatory cytokines, primarily IL-12 and IFN-γ. The purpose of the present study was to characterize Neospora caninum profilin, termed NcProfilin. Recombinant NcProfilin was purified by affinity chromatography, and used to prepare specific antisera to allow characterization of native NcProfilin antigen in N. caninum tachyzoites. By immunoblotting, recombinant NcProfilin is 22 kDa, and is similar in size to the respective 22 kDa native protein. Immunofluorescence and immunoelectron microscopy localized native NcProfilin to the apical end of N. caninum tachyzoites. Incubation of recombinant NcProfilin with spleen cells from BALB/c mice induced release of IFN-γ. Also, injection of BALB/c mice with purified rNcProfilin elicited a strong IFN-γ and IL-12 responses at 6 and 24 h after injection indicating that NcProfilin may be an important protein in regulation of cytokine responses to N. caninum.     

Nucleosome deposition and DNA methylation may participate in the recognition of premature termination codon in nonsense-mediated mRNA decay

In non-mammalian eukaryotes, an abnormally long 3′ untranslated region (UTR) is generally thought to be the definitive signal in the recognition of a premature termination codon (PTC) in nonsense-mediated mRNA decay (NMD). However, because the lengths of 3′ UTRs in normal mRNAs are widely distributed, “abnormally long” is hard to define. Distinct peaks of nucleosome deposition and DNA methylation have recently been found at coding region boundaries. We propose that nucleosomes and DNA methylation just upstream of a normal stop codon are ideal indicators for the position of a normal stop codon and may thus serve as signals in PTC recognition.     

Phylogenetic analysis and evolution of aromatic amino acid hydroxylase

A study was performed to investigate the phylogenetic relationship among AAAH members and to statistically evaluate sequence conservation and functional divergence. In total, 161 genes were identified from 103 species. Phylogenetic analysis showed that well-conserved subfamilies exist. Exon-intron structure analysis showed that the gene structures of AAAH were highly conserved across some different lineage species, while some species-specific introns were also found. The dynamic distribution of ACT domain suggested one gene fusion event has occurred in eukaryota. Significant functional divergence was found between some subgroups. Analysis of the site-specific profiles revealed critical amino acid residues for functional divergence. This study highlights the molecular evolution of this family and may provide a starting point for further experimental verifications.