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International audience; The Sã o Paulo Metropolitan Area (SPMA) is a megacity with 20 million people and over 8 million vehicles. Over the past decade a large increase in biofuel usage, more notably ethanol by light-duty vehicles, has made Brazil, and in particular Sã o Paulo, a unique case worldwide. This study presents the first assessment of emission ratios of a selected group of volatile organic compounds (VOCs) relative to carbon monoxide (CO) under ambient conditions. The VOCs studied here include aromatics such as benzene (1.03 pptv/ ppbv CO), toluene (3.10 pptv/ppbv CO) and Oxygenated VOCs such as methanol (5.39 pptv/ppbv CO), acetaldehyde (3.93 pptv/ppbv CO), acetone (3.59 pptv/ppbv CO), methyl ethyl ketone (1.42 pptv/ppbv CO), and others. Despite the specificity of the fuel composition, emission ratios were in surprisingly close agreement with other megacities in Europe or in North America. Such results include species whose emission factors have been previously reported to decline (e.g., benzene) or increase (e.g., acetaldehyde) with ethanol usage. Furthermore, diurnal profiles and temperature analysis aid separating the primary anthropogenic, secondary or biogenic components of the species studied here. This study shows that a significant fraction of ethanol in gasoline blends does not result in a well-defined trend in VOC emission profile and certainly motivates further studies.

International audience; Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80-90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines.

International audience; The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.