Development of High-Throughput Phenotyping of Metagenomic Clones from the Human Gut Microbiome for Modulation of Eukaryotic Cell Growth

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.     

Co-Circulation of Dengue Virus Type 3 Genotypes in Vientiane Capital,Lao PDR

During the 2012 epidemic of dengue in Vientiane capital, Lao PDR, a major serotype switch from dengue 1 to 3 was observed. A molecular epidemiology study demonstrated that dengue 3 remained the predominant serotype in 2013, but also revealed the co-circulation of two genotypes, supporting the hypothesis of multiple geographic origins of dengue 3 strains circulating in Vientiane capital.     

Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage

By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.     

A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library

The construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16S rDNA sequences has been used for over two decades to examine bacterial biodiversity. Here, we show that the analysis of random sequence reads (RSRs) instead of 16S is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. We generated 10 010 RSRs from a metagenomic library of microorganisms found in human faecal samples. Then searched them using the program BLASTN against a prokaryotic sequence database to assign a taxon to each RSR. The results were compared with those obtained by screening and analysing the clones containing 16S rDNA sequences in the whole library. We found that the biodiversity observed by RSR analysis is consistent with that obtained by 16S rDNA. We also show that RSRs are suitable to compare the biodiversity between different metagenomic libraries. RSRs can thus provide a good estimate of the biodiversity of a metagenomic library and, as an alternative to 16S, this approach is both faster and cheaper.     

Genomic choice of codons in 16 microbial species

We study the codon usage over whole set of ORFs of 16 unicellular microbial species: eight archaebacteria, seven eubacteria, and one eukarya. We first try to define, for each species, the neutral expected codon usage to better approach subsequently the influence of selection. Overlapping triplets counted from the complete DNA genomic sequence and mean amino acid composition of ORFs allow us to build satisfying expected codon usage for each species. Within species deviation from this neutral model is then studied through Correspondence Analysis and characterization with bias index, N(C)' (effective number of codons reported to neutral model). Our results are compared to previously published ones for three species and let appear good agreement in spite of very different methods. We thus propose set of codons probably preferred by selection for nine other species. In the four last species, no clear preference can be evidenced. Finally, we characterize variation of codon usage over functional categories. We propose that the high degree of bias of proteins involved in translation, ribosomal structure and biogenesis has a positive influence on overexpression of the corresponding genes under optimum growth conditions and is a negative regulator of the same genes when amino acids become limited resources.     

Susceptibility of human herpesvirus 6 to antiviral compounds by flow cytometry analysis

BACKGROUND: The emergence of human herpesvirus 6 (HHV-6) as a human pathogen led to the possibility of specific therapy against HHV-6 and the development of standardized susceptibility assays of HHV-6 to antivirals. METHODS: We have developed a flow cytometry method to analyze the multiplication of the HST strain of human herpesvirus 6 (HHV-6) variant B in vitro using monoclonal antibodies specific to virus proteins. This method was subsequently used to determine the sensitivity of HST multiplication in MT4 cells to four antiviral compounds of three different classes: acyclovir (ACV) and ganciclovir (GCV), two acyclic guanosine analogs; cedofovir (CDV), an acyclic nucleoside phosphonate; and phosphonoformic acid (PFA), a pyrophosphate analog. RESULTS: The 50% inhibitory concentrations (IC(50)) of ACV, GCV, CDV, and PFA determined by flow cytometry assay were 25.3, 6.4, 0.95, and 6.0 microM, respectively (5.7, 1.6, 0.3, and 1.8 microg/ml, respectively). These data together with the results of cytotoxicity assays confirmed the high efficiency and selectivity of CDV and PFA against HHV-6 B in vitro, suggested by previous results. CONCLUSIONS: Our flow cytometric assay appeared as a reproducible specific method to characterize HHV-6 susceptibility to antiviral compounds. It can be considered as a convenient alternative to the other immunologic and DNA hybridization assays used for that purpose.     

Using Plant Functional Traits and Phylogenies to Understand Patterns of Plant Community Assembly in a Seasonal Tropical Forest in Lao PDR

Plant functional traits reflect different evolutionary responses to environmental variation, and among extant species determine the outcomes of interactions between plants and their environment, including other plant species. Thus, combining phylogenetic and trait-based information can be a powerful approach for understanding community assembly processes across a range of spatial scales. We used this approach to investigate tree community composition at Phou Khao Khouay National Park (18°14’-18°32’N; 102°38’- 102°59’E), Laos, where several distinct forest types occur in close proximity. The aim of our study was to examine patterns of plant community assembly across the strong environmental gradients evident at our site. We hypothesized that differences in tree community composition were being driven by an underlying gradient in soil conditions. Thus, we predicted that environmental filtering would predominate at the site and that the filtering would be strongest on sandier soil with low pH, as these are the conditions least favorable to plant growth. We surveyed eleven 0.25 ha (50x50 m) plots for all trees above 10 cm dbh (1221 individual trees, including 47 families, 70 genera and 123 species) and sampled soils in each plot. For each species in the community, we measured 11 commonly studied plant functional traits covering both the leaf and wood economic spectrum traits and we reconstructed a phylogenetic tree for 115 of the species in the community using rbcL and matK sequences downloaded from Genebank (other species were not available). Finally we compared the distribution of trait values and species at two scales (among plots and 10x10m subplots) to examine trait and phylogenetic community structures. Although there was strong evidence that an underlying soil gradient was determining patterns of species composition at the site, our results did not support the hypothesis that the environmental filtering dominated community assembly processes. For the measured plant functional traits there was no consistent pattern of trait dispersion across the site, either when traits were considered individually or when combined in a multivariate analysis. However, there was a significant correlation between the degree of phylogenetic dispersion and the first principle component axis (PCA1) for the soil parameters. Moreover, the more phylogenetically clustered plots were on sandier soils with lower pH. Hence, we suggest that the community assembly processes across our site may reflect the influence of more conserved traits that we did not measure. Nevertheless, our results are equivocal and other interpretations are possible. Our study illustrates some difficulties in combining trait and phylogenetic approaches that may result from the complexities of integrating spatial and evolutionary processes that vary at different scales.     

Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.