Positive selection and evolution of dengue type-3 virus in the Indian subcontinent.

BACKGROUND: Dengue virus infection has recently taken endemic proportions in India with dengu type-3 (DEN-3) as a predominant serotype. In this study, we carried out the selection pressure analysis of three critical immunogenic regions of DEN-3. Phylogenetic analysis was then carried out on the positively selected genomic region in the DEN-3 virus strains isolated in the Indian subcontinent over a time span of 25 yr (1984-2008). Bayesian Markov chain Monte Carlo (MCMC) calculation of the substitution rate was carried out for the DEN-3 genotype-III sequences. METHODS: Sequences corresponding to the C-prM, E-NS1 and NS1 sequence regions of DEN-3 strains were taken for the positive selection analysis. The C-prM junction sequences were then used to construct a maximum likelihood (ML) phylogenetic tree. Substitution rates were also calculated under various models of population growth. RESULTS: It was found that codon 86, corresponding to a conserved arginine residue in a crucial T-cell epitope of the C-protein was under significant positive selection. The K86R substitution was found to exist in almost all the Indian strains isolated after 2004. The ML tree constructed from the C-prM junction sequences indicated that strains from the 2006 dengue incidences in Delhi, namely: 04/03/del2006, 05/03/del2006, and 06/03/del2006 were the most rapidly evolving. Substitution rates of a DEN-3 genotype-III sequences from the Indian subcontinent were found to be ~3.0 times higher than those reported from other parts of the world. CONCLUSION: Positive selection in the codon corresponding to R86 of the highly conserved surface C-protein is important in view of its occurrence in a T-cell epitope as well as its strict conservation in all the DEN strains. Phylogenetic analysis of the C-prM junction sequences showed that three strains of 2006 are rapidly evolving. These results were also supported by calculations of the substitution rates. Their significance in the expansion of viral epidemics requires to be investigated.

Purification of transcripts and metabolites from Drosophila heads.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.