A genetic screening for the tumour suppressive anticancer genes

Here, using a gain of function genetic screen approach I investigated 377 human apoptosis inducer genes that were previously isolated in HEK293T cells, and performed several rounds of genetic screens to isolate 22 human anticancer genes that only induce cell death in transformed but not normal cells. Later, using the genetically well-characterised cell line HEK293T as well as establishing transformed variants overexpressing individual oncogene, mutated genes or by knocking down tumour suppressor genes, I performed a separate genetic ‘synthetic lethal screen’ to identify a number of transformation scenarios in which these anticancer genes are targeted. Of these 22 anticancer genes, 16 showed a pronounced cell death inducing effect against c-Myc upregulation. c-Myc overexpressing cells were characterised to show phenotypic differences compared to their normal counterpart. On further validation, c-Myc specificity for these 16 genes was confirmed in a different cellular background in which some of these genes show inverse correlation to c-Myc expression. The last part of my project is to explore the molecular mechanism by which these 16 c-Myc specific anticancer genes induced cell death in c-Myc overexpressing cells. For the gene 1A3 (TMEFF2), my data indicated that it inhibited NF-B activity and CDK5 by inducing c-Myc specific cell death. In addition, 3 other genes 3B6, 4G3 and 4G4 also showed to induce c-Myc specific cell death through inhibition of FOXK2 in c-Myc overexpressing cells. In conclusion, I demonstrate that this ‘gain of function’ genetic screening strategy is useful for the isolation of tumour suppressor genes, but the establishment of their potential ‘anticancer’ functions would require further understanding of their molecular modes of action.Open Acces

Gain-of-function mutation of tristetraprolin impairs negative feedback control of macrophages in vitro yet has overwhelmingly anti-inflammatory consequences in vivo

The mRNA-destabilizing factor tristetraprolin (TTP) binds in a sequencespecific manner to the 3= untranslated regions of many proinflammatory mRNAs and recruits complexes of nucleases to promote rapid mRNA turnover. Mice lacking TTP develop a severe, spontaneous inflammatory syndrome characterized by the overexpression of tumor necrosis factor and other inflammatory mediators. However, TTP also employs the same mechanism to inhibit the expression of the potent antiinflammatory cytokine interleukin 10 (IL-10). Perturbation of TTP function may therefore have mixed effects on inflammatory responses, either increasing or decreasing the expression of proinflammatory factors via direct or indirect mechanisms. We recently described a knock-in mouse strain in which the substitution of 2 amino acids of the endogenous TTP protein renders it constitutively active as an mRNA-destabilizing factor. Here we investigate the impact on the IL-10-mediated anti-inflammatory response. It is shown that the gain-of-function mutation of TTP impairs IL-10-mediated negative feedback control of macrophage function in vitro. However, the in vivo effects of TTP mutation are uniformly anti-inflammatory despite the decreased expression of IL-10

Dominant suppression of inflammation via targeted mutation of the mRNA destabilizing protein tristetraprolin

In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies

Determining the Conditions That Lead to the Self-Extinguished and Self-Sustained Smoldering Combustion of Wood

To improve our understanding of flaming, smoldering, or self-extinction in the burning of wood, it is necessary to quantify the conditions that lead to self-extinguished and self-sustained smoldering combustion. Experiments were performed in a cone calorimeter under an external irradiation of 10 to 25 kW/m2 to analyze the temperature and mass loss of self-extinguished and self-sustained smoldering. The smoldering front depth was the significant parameter used to capture the smoldering characteristic, and it was defined as the axial thickness that reaches the smoldering characteristic temperature. The critical smoldering front depth of self-extinguished smoldering was lower than 10–15 mm for 30 mm thick wood at 15.5 kW/m2 irradiation. This critical depth decreased with the increase in heat flux, from 26.5 ± 1.5 mm at 10 kW/m2 to 11 ± 1 mm at 25 kW/m2. A simple theoretical analysis is proposed to explain the smoldering thickness threshold of self-sustained smoldering propagation based on the local heat balance. The equation predicts that the critical depth decreases as the heat flux increases, from 23.9 mm at 8 kW/m2 to 7.3 mm at 25 kW/m2. The predicted critical depth and heating duration were consistent with the experimental results. This study proposes a feasible parameter to help understand the threshold of smoldering propagation and the development of biomass burners

Research on Chinese Fire Station Optimal Location Model Based on Fire Risk Statistics: Case Study in Shanghai

With the rapid development of urbanization in China, the gap between urban and rural areas is decreasing. The traditional approach of constructing fire stations based on urban built-up areas is no longer suitable for the needs of modern fire rescue. Therefore, a comprehensive fire station location model is proposed based on fire risk assessment. This method divides the protected area units based on the urban road network. By evaluating different regions based on spatial position, land attributes, population density, floor area ratio, and fire incident indicators, the fire rescue risk levels and categorize regions into four risk levels are assessed. Corresponding response times were determined, and an objective model was developed to maximize the coverage area for fire response. The Baidu API was utilized to accurately calculate driving distances and times, and Gurobi optimization software was used to solve the model. Taking Shanghai as an example, the fire station location and layout from two perspectives—re-planning based on overall station placement and re-planning based on existing stations—were analyzed. The results suggest that constructing around 150 fire stations in Shanghai would effectively meet the fire rescue needs, which aligns with the actual situation in Shanghai and demonstrates the strong applicability of this model. This approach enables the meeting of new demand for fire station construction due to the significant increase in the coverage area while effectively utilizing firefighting resources

Selectin Ligand Sialyl-Lewis x Antigen Drives Metastasis of Hormone-Dependent Breast Cancers

Abstract The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLex; FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLex expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLex in ER-positive tumors was correlated with metastasis to the bone where sLex receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLex but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLex and E-selectin–dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)–dependent manner that was independent of sLex expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLex expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLex and HS glycosaminoglycans. Cancer Res; 71(24); 7683–93. ©2011 AACR.</jats:p