Comparative genomics of biotechnologically important yeasts

Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation

Safety assessment of excipients (SAFE) for orally inhaled drug products.

The development of new orally inhaled drug products requires the demonstration of safety which must be proven in animal experiments. New in vitro methods may replace, or at least reduce, these animal experiments provided they are able to correctly predict the safety or eventual toxicity in humans. However, the challenge is to link human in vitro data to human in vivo data. We here present a new approach to the safety assessment of excipients (SAFE) for pulmonary drug delivery. The SAFE model is based on a dose response curve of 23 excipients tested on the human pulmonary epithelial cell lines A549 and Calu-3. The resulting in vitro IC50 values were correlated with the FDA-approved concentration in pharmaceutical products for either pulmonary (if available) or parenteral administration. Setting a threshold of 0.1% (1 mg/mL) for either value yielded four safety classes, allowed to link IC50 data as measured on human cell cultures in vitro with the concentrations of the same compounds in FDA-approved drug products. The necessary in vitro data for novel excipients can be easily generated while the SAFE approach allows putting them in the context for eventual use in human pulmonary drug products. Excipients, that are most likely not safe for use in humans, can be early excluded from further pharmaceutical development. The SAFE approach helps thus to avoid unnecessary animal experiments

Expression of epithelial and mesenchymal markers in <i>Sca-1⁺/CD34^+,⁻</i> cells.

<p><b>Panel A:</b> The expression of epithelial and mesenchymal transcripts was tested by analytical PCR and is illustrated in a hierarchical cluster heatmap. The analysis shows that the majority of <i>Sca-1⁺/CD34⁺</i> cells (light grey) show similar marker expression as <i>Sca-1^−/CD34⁺</i> cells (white), while all <i>Sca-1⁺/CD34</i>⁻ cells (dark grey) are located in the second branch. Red squares indicate specific bands in analytical PCR, black squares indicate negative PCR results. <b>Panel B:</b> FACS analysis reveals EpCAM⁺/Pdgfrα⁺ subpopulation within Sca-1⁺/CD34^−/CD31^−/CD45⁻ cells. For each of 5 mice, 5×10⁶ murine lung cells were isolated from lung explants and stained with antibodies directed against Sca-1, CD34, CD31, CD45, Pdgfrα and Epcam. While Sca-1⁺/CD34⁻ cells consistently showed Epcam expression, Pdgfrα expression was predominantly found in Sca-1⁺/CD34⁺ cells. However, Sca-1⁺/CD34^−/Epcam⁺ cells could be divided in two major subpopulations defined by Pdgfrα expression. Relative quantification is given for corresponding selected subpopulation as indicated by arrows. <b>Panel C:</b> Scatter plots of the detected cell populations for mouse 5, only.</p

PCR results of corresponding transcripts in Sca-1+/CD31- and cells CD34+/CD45- cells.

<p>PCR results of corresponding transcripts in Sca-1+/CD31- and cells CD34+/CD45- cells.</p

Distribution of PCR-based <i>Sca-1/CD34</i> expression in isolated putative BASCs.

<p>Distribution of PCR-based <i>Sca-1/CD34</i> expression in isolated putative BASCs.</p