A single positively selected West Nile viral mutation confers increased virogenesis in American crows

West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the well-described epidemiological patterns of WNV in North America, the basis for the emergence of WNV-associated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis

Cancer cells are typically characterized by apoptosis deficiency. In order to investigate a possible role for the anti-apoptotic livin gene in renal cell cancer (RCC), we analyzed its expression in tumor tissue samples and in RCC-derived cell lines. In addition, we studied the contribution of livin to the apoptotic resistance of RCC cells by RNA interference (RNAi). Livin gene expression was detected in a significant portion of RCC tumor tissue specimens (13/14, 92.9%) and tumor-derived cell lines (12/15, 80.0%). Moreover, targeted inhibition of livin by RNAi markedly sensitized RCC cells towards proapoptotic stimuli, such as UV irradiation or the chemotherapeutic drugs etoposide, 5-fluorouracil, and vinblastine. These effects were specific for livin expressing tumor cells. We conclude that livin can contribute significantly to the apoptosis resistance of RCC cells. Targeted inhibition of livin could represent a novel therapeutic strategy to increase the sensitivity of renal cancers towards pro-apoptotic agents. Received 30 November 2006; received after revision 22 February 2007; accepted 20 March 2007     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

Nest architecture in polydomous grass-cutting ants (Acromyrmex balzani)

Acromyrmex balzani is a grass-cutting ant species frequently found in Cerrado areas. However, little is known about the architecture of the polydomous nests of this ant. Fifteen A. balzani nests located in a cerrado region in Botucatu, São Paulo, Brazil, were studied. The polydomous nests were studied in three ways. First, we investigated the architecture in nests moulded with cement and without moulding. Second, we performed an aggressiveness test among workers in different subnests and nests. Third, we excavated the nest and collected the colony to measure the population, verifying the existence or not of a queen in all nests. A cement mould was made of seven nests to permit better visualization of internal structures such as chambers and tunnels. Eight nests were excavated without moulding and white neutral talc was used to highlight the parts of the nests. After excavation, the depth and dimensions (length, width and height) of the chambers were measured. The results showed that the nests had a single entrance hole whose structure consisted of straw and other plant residues in winter. Mounds of loose soil, if present, were found 6–48 cm from the hole. The number of chambers containing fungus ranged from one to five, with the first being found a few centimetres beneath the ground surface (4 cm) and the last up to a maximum depth of 160 cm. The length of the tunnels ranged from 12 to 28 cm. These tunnels were built in a vertical or inclined position, leading to the chambers. No waste chambers were found, with the waste being deposited externally. Additionally, the polydomous nests contained one to eight subnests. In the aggressiveness test, when concolonial workers were confronted, no aggressiveness was observed. In contrast, when allocolonial workers were confronted, there was a high incidence of aggression among them. Excavation of polydomous nests showed only one queen for each polydomous nest, i.e. subnests with a single queen. Our study contributes to a better understanding of the so far unknown nest architecture of the polydomous grass-cutting ant A. balzani.     

脱硫催化剂的酸法制备及性能研究

对用酸法进行催化剂制备，酸法制备的最佳镁铝比，分散介质的优化，引入对脱催化剂的氧化性能和还原性能的影响。以及实验室的还原温度等诸方面进行了研究。