Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [¹³C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.     

Identification of major proteins secreted by Cryptococcus neoformans

The characterization of proteins secreted by Cryptococcus neoformans is of relevance to the identification of vaccine candidates, because concentrated supernatants from the fungus have been shown to be immunoprotective in previous studies. After fractionation of supernatants by anion exchange chromatography and preparative electrophoresis, we obtained the N-terminal amino acid sequences of 13 major proteins. Using a C. neoformans nucleotide database, we were able to clone and sequence the ORFs coding for 12 of these proteins. Some of the genes are identical to previously described ones, while six encode novel proteins, including four putative mannoproteins. The molecular characterization of these and other secreted products may provide useful information in the development of immune-based strategies to control cryptococcosis.     

Novel tricyclic antagonists of the prostaglandin D2 receptor DP2 with efficacy in a murine model of allergic rhinitis

The synthesis of a series of tricyclic antagonists for the prostaglandin D₂ receptor DP2 (CRTH2) is disclosed. The activities of the compounds were evaluated in a human DP2 binding assay and a human whole blood eosinophil shape change assay. Potential metabolic liabilities of the compounds were addressed through in vitro CYP studies. The lead compound was demonstrated to have efficacy in a mouse model of allergic rhinitis following oral dosing.     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

Broussonetia papyrifera controls nutrient return to soil to facilitate its invasion in a tropical forest of Ghana

Aims Non-native invasive plants can alter soil chemistry through litter production and decomposition to facilitate their invasion. However, the important roles of these underlying processes in plant invasion remain poorly understood, particularly in tropical forest ecosystems. Here, we compared litter production, quality and decomposition of two invasive species (Broussonetia papyriferaandCedrela odorata) and two co-occurring native species (CeltismildbraediiandFuntumia elastica), and soil properties under them to elucidate their roles in the invasion of a tropical forest in Ghana.     

Large Rainfall Pulses Control Litter Decomposition in a Tropical Dry Forest: Evidence from an 8-Year Study

We investigated the influence of rainfall attributes on litter decomposition over an 8-year period in a well-preserved tropical dry forest ecosystem in western Mexico. We examined the relationship between the size and number of rainfall events and rainy-season litter decomposition rates and determined if this relationship varied along a landscape gradient. A mass balance approach was used to estimate decomposition rate in four permanent 2,400 m² plots located in two small watersheds. Watershed I included three plots in different landscape positions (upper, middle, and lower) in the elevation gradient, whereas Watershed IV included one plot in the middle position. Surface litter C mass was lower in the rainy than in the dry season in all plots in response to seasonal fluctuations in rainfall. The frequency of small (≤5 mm) and medium (5.1–9.9 mm) size rainfall events largely did not correlate with litter decomposition, but the frequency of large events (≥10 mm) had a positive correlation with decomposition rates (P < 0.05), except in plot IV (P < 0.1). Decomposition rates were similar among plots at the different landscape positions within Watershed I (P > 0.05). The relevance of large rainfall events (≥10 mm) in rainy-season litter decomposition suggests that changes in the precipitation regime which alter the frequency of these rainfall pulses or increase their variability would affect the vulnerability of the litter C and nutrient pools to extreme events.     

Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.