Development of tools to automate quantitative analysis of radiation damage in SAXS experiments

Biological small-angle X-ray scattering (SAXS) is an increasingly popular technique used to obtain nanoscale structural information on macromolecules in solution. However, radiation damage to the samples limits the amount of useful data that can be collected from a single sample. In contrast to the extensive analytical resources available for macromolecular crystallography (MX), there are relatively few tools to quantitate radiation damage for SAXS, some of which require a significant level of manual characterization, with the potential of leading to conflicting results from different studies. Here, computational tools have been developed to automate and standardize radiation damage analysis for SAXS data. RADDOSE-3D, a dose calculation software utility originally written for MX experiments, has been extended to account for the cylindrical geometry of the capillary tube, the liquid composition of the sample and the attenuation of the beam by the capillary material to allow doses to be calculated for many SAXS experiments. Furthermore, a library has been written to visualize and explore the pairwise similarity of frames. The calculated dose for the frame at which three subsequent frames are determined to be dissimilar is defined as the radiation damage onset threshold (RDOT). Analysis of RDOTs has been used to compare the efficacy of radioprotectant compounds to extend the useful lifetime of SAXS samples. Comparison of the RDOTs shows that, for radioprotectant compounds at 5 and 10 mM concentration, glycerol is the most effective compound. However, at 1 and 2 mM concentrations, di­thio­threitol (DTT) appears to be most effective. Our newly developed visualization library contains methods that highlight the unusual radiation damage results given by SAXS data collected using higher concentrations of DTT: these observations should pave the way to the development of more sophisticated frame merging strategies

Adaptive Traits Are Maintained on Steep Selective Gradients despite Gene Flow and Hybridization in the Intertidal Zone

Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal) was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain the 3 distinct genetic and morphological taxa within their preferred vertical distribution ranges. On the strength of distributional, genetic, physiological and morphological differences, we propose elevation of F. spiralis var. platycarpus from variety to species level, as F. guiryi

Nanometre resolution tracking of myosin-1b motility.

The movement produced by a small number of myosin molecular motors was measured with nanometre precision using single-molecule fluorescence localisation methods. The positional precision of the measurements was sufficient to reveal fluctuations in sliding velocity due to stochastic interactions between individual myosin motors and the actin filament. Dependence of sliding velocity upon filament length was measured and fluctuations in velocity were quantified by autocorrelation analysis. Optical tweezers-based nanometry was used to measure the myosin-1b step-size directly. The 10 nm power-stroke and its duty cycle ratio were consistent with values derived from in vitro sliding assays

A model of stereocilia adaptation based on single molecule mechanical studies of myosin I.

We have used an optical tweezers-based apparatus to perform single molecule mechanical experiments using the unconventional myosins, Myo1b and Myo1c. The single-headed nature and slow ATPase kinetics of these myosins make them ideal for detailed studies of the molecular mechanism of force generation by acto-myosin. Myo1c exhibits several features that have not been seen using fast skeletal muscle myosin II. (i) The working stroke occurs in two, distinct phases, producing an initial 3 nm and then a further 1.5 nm of movement. (ii) Two types of binding interaction were observed: short-lived ATP-independent binding events that produced no movement and longer-lived, ATP-dependent events that produced a full working stroke. The stiffness of both types of interaction was similar. (iii) In a new type of experiment, using feedback to apply controlled displacements to a single acto-myosin cross-bridge, we found abrupt changes in force during attachment of the acto-Myo1b cross-bridge, a result that is consistent with the classical 'T2' behaviour of single muscle fibres. Given that these myosins might exhibit the classical T2 behaviour, we propose a new model to explain the slow phase of sensory adaptation of the hair cells of the inner ear

Visualizing helicases unwinding DNA at the single molecule level.

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes

Occupational image, organisational image and identity in dirty work: Intersections of organisational efforts and media accounts

This article proposes that media representations of an occupational category may intersect with organizations’ efforts to construct a positive organizational identity and image. We fuse three streams of literature namely, organizational identity and image, media and the social construction of reality, and dirty work to extend extant literature on organizational identity and image. Attention is drawn to occupational image as the position of an occupational category in society. We contend that occupational image is likely to influence the decisions and actions taken by organizations and its members, in particular when the occupation is central to the organization’s mission. Occupational image is partly informed by the media. We analyse one year of media coverage of a dirty work occupation, specifically exotic dancing, and identify various ways in which the media portrays the exotic dancing occupation and the organizations providing these services. We focus upon two of these categories, namely Public (dis) Order and Art and Entertainment. We also draw upon a variety of data from one organization, For Your Eyes Only, to explore how organizational efforts to construct a positive organizational identity (based upon professionalism and legitimacy) and image (based upon fantasy, exclusivity and high quality service) intersect these media representations