A rapid method to assess reactive oxygen species in yeast using H2DCF-DA

A protocol to efficiently assess Reactive Oxygen Species (ROS) levels in yeast cells using H2DCF-DA is described here. This method employs lithium acetate to permeate the cell wall, and thus, augments the release of the fluorescent product, dichlorofluorescein from the cells. This protocol obviates the need for both physical and enzymatic lysis methods that are arduous and time consuming. This method is simple, less time consuming and reproducible, especially while dealing with a large sample size. The lithium acetate method gave significantly reproducible and linear results (P < 0.0001), as compared with direct measurement (P = 0.0005), sonication (P = 0.1466) and bead beating (P = 0.0028).

Structural studies on tobacco streak virus coat protein: Insights into the pleomorphic nature of ilarviruses

Tobacco streak virus (TSV), the type member of Ilarvirus genus, is a major plant pathogen. TSV purified from infected plants consists of a ss-RNA genome encapsidated in spheroidal particles with diameters of 27, 30 and 33 nm constructed from multiple copies of a single species of coat protein (CP) subunits. Apart from protecting the viral genome, CPs of ilarviruses play several key roles in the life cycle of these viruses. Unlike the related bromo and cucumoviruses, ilarvirus particles are labile and pleomorphic, which has posed difficulties in their crystallization and structure determination. In the current study, a truncated TSV-CP was crystallized in two distinct forms and their structures were determined at resolutions of 2.4 angstrom and 2.1 angstrom, respectively. The core of TSV CP was found to possess the canonical beta-barrel jelly roll tertiary structure observed in several other viruses. Dimers of CP with swapped C-terminal arms (C-arm) were observed in both the crystal forms. The C-arm was found to be flexible and is likely to be responsible for the polymorphic and pleomorphic nature of TSV capsids. Consistent with this observation, mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses. This communication represents the first report on the structure of the CP from an ilarvirus. (C) 2015 Elsevier Inc. All rights reserved.