A method improving the accuracy of fluorescence recovery after photobleaching analysis

Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/noise ratio is low, when there are temporal fluctuations in the illumination, or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under nonideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within ∼1%, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with <10% error.     

Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.     

Leaf domatia reduce intraguild predation among predatory mites

1. Although theory suggests that intraguild predation destabilises food webs and may result in exclusion of species, empirical observations of food webs reveal that it is a common interaction. It has been proposed that habitat structure reduces the interaction strength of intraguild predation, thus facilitating the coexistence of species. 2. This was tested using acarodomatia, tiny structures on plant leaves, and predatory mites, which usually reside in these domatia. Sweet pepper plants (Capsicum annuum L.) were used, which possess domatia consisting of tufts of hair, and coffee plants (Coffea arabica L.) with pit‐shaped domatia. 3. On sweet pepper, the predatory mites Neoseiulus cucumeris Oudemans and Iphiseius degenerans Berl. feed on each other's juveniles. Larvae of each of the species were therefore used as intraguild prey with adult females of the other species as intraguild predators. On coffee, a similar set‐up was used, with larvae and adult females of Amblyseius herbicolus Chant and Iphiseiodes zuluagai Denmark & Muma as intraguild prey and intraguild predators, respectively. 4. Domatia on detached, isolated sweet pepper and coffee leaves were either closed with glue or left open, after which larvae and adult predators were released. As a control, larvae were released on leaves with open or closed domatia without an adult predator. 5. Survival of larvae was high in the absence of the adult (intraguild) predator. In the presence of the intraguild predator, survival was significantly higher on leaves with open domatia than on leaves with closed domatia. 6. This shows that even such tiny structures as plant domatia may significantly affect the interaction strength of intraguild predation.     

The circatidal rhythm of the estuarine gastropod Hydrobia ulvae (Gastropoda: Hydrobiidae)

Intertidal animals display a suite of cyclic behaviours that evolved as adaptations to the predictable cycle of inundation and exposure. In estuarine habitats, mud snails from the genus Hydrobia are among the most abundant grazers, and have received considerable attention with respect to the behavioural mechanisms mediating locomotion, dispersal, and feeding, although the nature of the control of these processes has remained elusive. In particular, it is not clear whether endogenous activity patterns are related to periodic changes of microphytobenthos biomass at the sediment surface, or whether they are timed to the tidal cycle at all. In the present study, we address the crawling activity of Hydrobia ulvae under constant conditions, as well as the effects of individual size and previous short‐term exposure to tides of different range, by recording immersed individual snails under constant dark conditions. We show that the species displays an overt circatidal pattern of crawling, with activity peaks around high water, and that the start of inundation may act as an entrainment agent of the rhythm. Moreover, the results obtained indicate that smaller snails display higher levels of activity, although neither the size nor previous in situ influence of tidal range has an effect on the period and on the amplitude of the rhythm. These findings suggest that fluctuations of microphytobenthos biomass are not a sufficiently strong selective pressure to have shaped locomotor activity in H. ulvae. Moreover, feeding of H. ulvae should take place mostly during high water and be independent of periodic fluctuations of microphytobenthos biomass at the surface of the sediment. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 439–450.     

A prospective study of delayed sleep phase syndrome in patients with severe resistant obsessive-compulsive disorder

There have been relatively few studies examining sleep in patients with obsessive-compulsive disorder (OCD) and these have produced contradictory findings. A recent retrospective study identified a possible association between OCD and a circadian rhythm sleep disorder known as delayed sleep phase syndrome (DSPS). Patients with this pattern of sleeping go to bed and get up much later than normal. They are unable to shift their sleep to an earlier time and, as a result, suffer considerable disruption to social and occupational functioning. In this study, we examined the sleep of patients with OCD prospectively. We aimed to establish the frequency of DSPS in this population and any associated clinical or demographic factors which might be implicated in its aetiology.     

Quantitative comparisons ofin situ microbial biodiversity by signature biomarker analysis

Microscopic examinations have convinced microbial ecologists that the culturable microbes recovered from environmental samples represent a tiny proportion of the extant microbiota. Methods for recovery and enzymatic amplification of nucleic acids from environmental samples have shown that a huge diversity existsin situ, far exceeding any expectations which were based on direct microscopy. It is now theoretically possible to extract, amplify and sequence all the nucleic acids from a community and thereby gain a comprehensive measure of the diversity as well as some insights into the phylogeny of the various elements within this community. Unfortunately, this analysis becomes economically prohibitive if applied to the multitude of niches in a single biome let alone to a diverse set of environments. It is also difficult to utilize PCR amplification on nucleic acids from some biomes because of coextracting enzymatic inhibitors. Signature biomarker analysis which potentially combines gene probe and lipid analysis on the same sample, can serve as a complement to massive environmental genome analysis in providing quantitative comparisons between microniches in the biome under study. This analysis can also give indications of the magnitude of differences in biodiversity in the blome as well as provide insight into the phenotypic activities of each community in a rapid and cost-effective manner. Applications of signature lipid biomarker analysis to define quantitatively the microbial viable biomass of portions of an Eastern USA deciduous forest, are presented.