LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

An improved histochemical method for measuring nitric oxide synthase activity

The previous quantitative histochemical method for measuring nitric oxide synthase (NOS) activity in tissue sections involved the loss of about 15 per cent of the NOS, presumably from the section into the reaction medium. Two changes are now described. The first is concerned with the preparation in the laboratory of the active reagent, lead ammonium citrate/acetate (LACA). The second change involves an improvement of the procedure for measuring NOS activity. The new method appears to retain all the measurable NOS activity inside the section. Copyright © 1999 John Wiley & Sons, Ltd.     

Employee Attitudes about the Impact of Visitation Dogs on a College Campus

Therapy and visitation dogs are becoming more common on college campuses to provide comfort and support to students, but little attention has been given to the concerns of faculty and staff who share space with the dogs in their workplaces. The purpose of this study was to assess the perceptions of faculty and staff with regard to both the benefits and the hazards (e.g., dander, bites, fleas) and risks associated with the presence of visitation dogs in their workplaces. One hundred and thirty-eight employees who worked in buildings with resident visitation dogs completed an online survey about their perceptions of the hazards and risks of the dogs and the effects of dogs on the wellbeing of both students and employees. In general, employees perceived that the dogs presented minimal risks, and most employees believed that they can reduce stress and provide comfort to students on campus. There were a few employees, however, who reported that the dogs did not improve the work environment and conferred no benefits to the staff or students. The findings of the present survey support the mostly positive attitudes that people have for dogs in the workplace, but they also highlight a potential challenge: accommodating individuals who believe very strongly that dogs do not belong in work environments.     

A study of candidate genes for day blindness in the standard wire haired dachshund

Background

A genetic study was performed to identify candidate genes associated with day blindness in the standard wire haired dachshund. Based on a literature review of diseases in dogs and human with phenotypes similar to day blindness, ten genes were selected and evaluated as potential candidate genes associated with day blindness in the breed.

Results

Three of the genes, CNGB3, CNGA3 and GNAT2, involved in cone degeneration and seven genes and loci, ABCA4, RDH5, CORD8, CORD9, RPGRIP1, GUCY2D and CRX, reported to be involved in cone-rod dystrophies were studied. Polymorphic markers at each of the candidate loci were studied in a family with 36 informative offspring. The study revealed a high frequency of recombinations between the candidate marker alleles and the disease.

Conclusion

Since all of the markers were at the exact position of the candidate loci, and several recombinations were detected for each of the loci, all ten genes were excluded as causal for this canine, early onset cone-rod dystrophy. The described markers may, however, be useful to screen other canine resource families segregating eye diseases for association to the ten genes.

     

NiFe hydrogenase active site biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha

Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF. Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1. Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis. Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R. eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process. With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro. Furthermore, HypC and HypD functioned as a unit. Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry. The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells. We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex. Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE. On the basis of these results, we propose a complete catalytic cycle for HypE. First, it is modified by HypF1, and then it can form a complex with HypC/HypD. This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE.     

Lack of DNA polymerase μ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence

The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ^?/? mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ^?/? MEFs, associated with high levels of the tumor-suppressor p16^INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ^?/? MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ^?/? MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.     

Field and climate‐based model for predicting the density of host‐seeking nymphal Ixodes scapularis,an important vector of tick‐borne disease agents in the eastern United States

Aim Ixodes scapularis is the most important vector of human tick‐borne pathogens in the United States, which include the agents of Lyme disease, human babesiosis and human anaplasmosis, among others. The density of host‐seeking I. scapularis nymphs is an important component of human risk for acquiring Borrelia burgdorferi, the aetiological agent of Lyme disease. In this study we used climate and field sampling data to generate a predictive map of the density of host‐seeking I. scapularis nymphs that can be used by the public, physicians and public health agencies to assist with the diagnosis and reporting of disease, and to better target disease prevention and control efforts. Location Eastern United States of America. Methods We sampled host‐seeking I. scapularis nymphs in 304 locations uniformly distributed east of the 100th meridian between 2004 and 2006. Between May and September, 1000 m² were drag sampled three to six times per site. We developed a zero‐inflated negative binomial model to predict the density of host‐seeking I. scapularis nymphs based on altitude, interpolated weather station and remotely sensed data. Results Variables that had the strongest relationship with nymphal density were altitude, monthly mean vapour pressure deficit and spatial autocorrelation. Forest fragmentation and soil texture were not predictive. The best‐fit model identified two main foci – the north‐east and upper Midwest – and predicted the presence and absence of I. scapularis nymphs with 82% accuracy, with 89% sensitivity and 82% specificity. Areas of concordance and discordance with previous studies were discussed. Areas with high predicted but low observed densities of host‐seeking nymphs were identified as potential expansion fronts. Main conclusions This model is unique in its extensive and unbiased field sampling effort, allowing for an accurate delineation of the density of host‐seeking I. scapularis nymphs, an important component of human risk of infection for B. burgdorferi and other I. scapularis‐borne pathogens.