Characterization of Bordetella holmesii isolates from patients with pertussis-like illness in The Netherlands

Bordetella holmesii is a recently described human pathogen mainly isolated from blood. However, in the US and Canada, B. holmesii has also been cultured from the nasopharynx of patients with pertussis-like symptoms. To the best of our knowledge, respiratory isolates from Europe have not been characterized. Here, we report the isolation and characterization of B. holmesii from Dutch patients with pertussis-like illness. Species determination was confirmed by 16S rRNA gene sequencing and detection by PCR of IS481 and bhoE, a gene not found in Bordetella pertussis but present in B. holmesii. Comparative genomic hybridization (CGH) with microarrays revealed that the Dutch isolates formed a cluster distinct from isolates from the US and UK suggesting a distinct population or an epidemiological relationship between the Dutch isolates. All isolates contained a locus involved in iron uptake, previously suggested to originate from B. pertussis. The causes for the apparent increase in the isolation of B. holmesii are discussed.     

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By coimmunoprecipitation we show that ICAP-1 and ROCK form complexes in cells and that ICAP-1 contains two binding sites for ROCK. In cells transfected with both ICAP-1 and ROCK, the proteins colocalized at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. ROCK was not found at these sites when ICAP-1 was not co-transfected, indicating that ICAP-1 translocated ROCK. In lamellipodia ICAP-1 and ROCK colocalized with endogenous beta1 integrins and this colocalization was also observed with the isolated ICAP-1 PTB domain. The plasma membrane localization of ROCK did not depend on beta1 integrin ligation or ROCK kinase activity, and in truncated ROCK proteins it required the presence of the ICAP-1-binding domain. To show that the interaction was direct, we measured fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) fused to ICAP-1 and yellow fluorescent protein (YFP) fused to ROCK. FRET was observed in lamellipodia in cells that were induced to spread. These results indicate that ICAP-1-mediated binding of ROCK to beta1 integrin serves to localize the ROCK-I kinase to both the leading edge and the trailing edge where ROCK affects cell migration.     

ENOD40 affects elongation growth in tobacco Bright Yellow-2 cells by alteration of ethylene biosynthesis kinetics

Plant developmental processes are controlled by co-ordinated action of phytohormones and plant genes encoding components of developmental response pathways. ENOD40 was identified as a candidate for such a plant factor with a regulatory role during nodulation. Although its mode of action is poorly understood, several lines of evidence suggest interaction with phytohormone response pathways. This hypothesis was investigated by analysing cytokinin-, auxin-, and ethylene-induced responses on cell growth and cell division in transgenic 35S:NtENOD40 Bright Yellow-2 (BY-2) tobacco cell suspensions. It was found that cell division frequency is controlled by the balance between cytokinin and auxin in wild-type cells and that this regulation is not affected in 35S:NtENOD40 lines. Elongation growth, on the other hand, is reduced upon overexpression of NtENOD40. Analysis of ethylene homeostasis shows that ethylene accumulation is accelerated in 35S:NtENOD40 lines. ENOD40 action can be counteracted by an ethylene perception blocker, indicating that ethylene is a negative regulator of elongation growth in 35S:NtENOD40 cells, and that the NtENOD40-induced response is mediated by alteration of ethylene biosynthesis kinetics.     

The use of radiotelemetry to assess the time needed to acclimatize guineapigs following several hours of ground transport

The objective of this study was to investigate the effect of ground transportation on guineapigs. Physiological parameters, i.e. heart rate (HR), body temperature (BT) and activity (ACT), were measured before and after transport, using previously implanted radiotelemetry transmitters. Body weight was measured before and after transport. After a postsurgical recovery period and data recording at the breeder's facility, the animals were transported for 2.25 h (Group 1) and for 7.5 h (Group 2) to a different animal facility. Data collection started immediately after arrival at the second animal facility. All parameters measured changed significantly after transport. These results suggest that a 10- to 12-day period is required for guineapigs to return to pre-transport levels of HR, BT and ACT.     

Direct Spatial Control of Epac1 by Cyclic AMP

Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.Cyclic AMP (cAMP) is an important second messenger that mediates many cellular hormone responses. It has become more and more appreciated that, along with the cAMP effector protein kinase A (PKA), Epac proteins also play pivotal roles in many cAMP-controlled processes, including insulin secretion (23, 39), cell adhesion (9, 17, 25, 49, 60), neurotransmitter release (22, 53, 63), heart function (13, 35, 54), and circadian rhythm (38). Epac1 and Epac2 are cAMP-dependent guanine nucleotide exchange factors (GEFs) for the small G proteins Rap1 and Rap2 (12, 24). They contain a regulatory region with one (Epac1) or two (Epac2) cAMP-binding domains, a Dishevelled, Egl-10, Pleckstrin (DEP) domain, and a catalytic region for GEF activity (11). The binding of cAMP is a prerequisite for catalytic activity in vitro and in vivo (11). Recently, the structures of both the inactive and active conformations of Epac2 were solved (51, 52). This revealed that in the inactive conformation, the regulatory region occludes the Rap binding site, which is relieved by a conformational change induced by cAMP binding.Like all G proteins of the Ras superfamily, Rap cycles between an inactive GDP-bound and active GTP-bound state in an equilibrium that is tightly regulated by specific GEFs and GTPase-activating proteins (GAPs). The GEF-induced dissociation of GDP results in the binding of the cellularly abundant GTP, whereas GAPs enhance the intrinsic GTPase activity of the G protein, thereby inducing the inactive GDP-bound state. Besides Epac, several other GEFs for Rap have been identified, including C3G, PDZ-GEF, and RasGRP, and these act downstream of different signaling pathways (7). Since Rap localizes to several membrane compartments, including the Golgi network, vesicular membranes, and the plasma membrane (PM) (2-4, 37, 42, 48), the spatial regulation of its activity is expected to be established by the differential distributions of its upstream GEFs, each activating distinct pools of Rap on specific intracellular locations.Similarly to Rap, Epac1 also is observed at many locations in the cell, including the cytosol, the nucleus, the nuclear envelope, endomembranes, and the PM (5, 11, 14, 21, 29, 47). These various locations may reflect the many different functions assigned to Epac1, such as the regulation of cell adhesion, cell junction formation, secretion, the regulation of DNA-dependent protein kinase by nuclear Epac1, and the regulation of the Na⁺/H⁺ exchanger NHE3 at the brush borders of kidney epithelium (19, 21, 26). Apparently, specific anchors are responsible for this spatial regulation of Epac1. Indeed, Epac1 was found to associate with phosphodiesterase 4 (PDE4) in a complex with mAKAP in cardiomyocytes (13), with MAP-LC bound to microtubules (62), and with Ezrin at the brush borders of polarized cells (M. Gloerich, J. Zhao, and J. L. Bos, unpublished data).In this study, we report the unexpected observation that, in addition to the temporal control of Epac1 activity, cAMP also induces the translocation of Epac1 toward the plasma membrane. Using confocal fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy, and fluorescence resonance energy transfer (FRET)-based assays for high spatial and temporal resolution, we observed that the translocation of Epac1 is immediate and that Epac1 approaches the PM to within ∼7 nm. In line with this, Epac1-induced Rap activation was registered predominantly on this compartment. Epac1 translocation results directly from the cAMP-induced conformational change and depends on the integrity of its DEP domain. We further show that Epac1 translocation is a prerequisite for cAMP-induced Rap activation at the PM and enhances Rap-mediated cell adhesion. Thus, cAMP exerts dual regulation on Epac1 for the activation of Rap, controlling both its GEF activity and targeting to the PM.     

FLUCTUATING SELECTION AND THE MAINTENANCE OF INDIVIDUAL AND SEX‐SPECIFIC DIET SPECIALIZATION IN FREE‐LIVING OYSTERCATCHERS

Fluctuating and disruptive selection are important mechanisms for maintaining intrapopulation trait variation. Nonetheless, few field studies quantify selection pressures over long periods and identify what causes them to fluctuate. Diet specialists in oystercatchers differ in short‐term payoffs (intake), but their long‐term payoffs are hypothesized to be condition dependent. We test whether phenotypic selection on diet specialization fluctuates between years due to the frequency of specialists, competitor density, prey abundance, and environmental conditions. Short‐term payoffs proved to be poor predictors of long‐term fitness payoffs of specialization. Sex‐differences in diet specialization were maintained by opposing directional fecundity and viability selection between the sexes. Contrasting other studies, selection on individual diet specialization was neither negative frequency‐ or density‐dependent nor dependent on prey abundance. Notwithstanding, viability selection fluctuated strongly (stabilizing?disruptive) over the 26‐year study period: slightly favoring generalists in most years, but strongly disfavoring generalists in rare harsh winters, suggesting generalists cannot cope with extreme conditions. Although selection fluctuated, mean selection on specialists was weak, which can explain how individual specialization can persist over long periods. Because rare events can dramatically affect long‐term selective landscapes, more care should be taken to match the timescale of evolutionary studies to the temporal variability of critical environmental conditions.     

Neurotoxicity of Alzheimer's disease A�� peptides is induced by small changes in the A��42 to A��40 ratio

The amyloid peptides Aβ₄₀ and Aβ₄₂ of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ₄₂ seems more pathogenic than Aβ₄₀, the reason for this is not well understood. We show here that small alterations in the Aβ₄₂:Aβ₄₀ ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ₄₂:Aβ₄₀ ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.     

Reduced blood parasite prevalence with age in the Seychelles Warbler: selective mortality or suppression of infection?

Avian malaria can affect survival and reproduction of their hosts. Two patterns commonly observed in birds are that females have a higher prevalence of malaria than do males and that prevalence decreases with age. The mechanisms behind these patterns remain unclear. However, most studies on blood parasite infections are based on cross-sectional analyses of prevalence, ignoring malaria related mortality and individual changes in infection. Here, we analyse both within-individual changes in malaria prevalence and long-term survival consequences of infection in the Seychelles Warbler (Acrocephalus sechellensis). Adults were less likely to be infected than juveniles but, contrary to broad patterns previously reported in birds, females were less likely to be infected than males. We show by screening individual birds in two subsequent years that the decline with age is a result both of individual suppression of infection and selective mortality. Birds that were infected early in life had a lower survival rate compared to uninfected birds, but among those that survived to be screened twice the proportion of infected birds had also decreased. Uninfected birds did not become infected later in life. Males were found to be more infected than females in this species possibly because, unlike most birds, males are the dispersing sex and the cost of dispersal may have to be traded against immunity. Infected males took longer to suppress their infection than did females. We conclude that these infections are indeed costly, and that age-related patterns in blood parasite prevalence are influenced both by suppression and selective mortality.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Optimal foraging on perilous prey: risk of bill damage reduces optimal prey size in oystercatchers

Intake rate maximization alone is not always sufficient in explainingprey size selection in predators. For example, bivalve-feedingoystercatchers regularly select smaller prey than expected ifthey aimed to maximize their intake rate. It has been proposedthat to these birds large prey are "risky," in the sense thatbirds may damage their bills when feeding on large bivalves.Large bivalves yield more energy, but according to this hypothesisthis is achieved at the expense of energy yield in the longterm when (1) the risk of bill damage increases with prey sizeand (2) foraging with a damaged bill is less effective. In accordancewith this hypothesis, we show that captive oystercatchers feedingon large cockles experienced a high probability of bill tipdamage, while bill damage was absent when cockles were small.Moreover, among free-living oystercatchers the prevalence ofbill damage was correlated with mean cockle size near the capturesite, and the data on captive birds fit in this pattern. Foodintake of captive oystercatchers feeding exclusively on cockleswas reduced by 23% after bill damage, and free-living birdswith damaged bills had 14 g lower mass. Because lower body masswas associated with higher mortality probability, these resultsindicate long-term costs associated with feeding on large cockles.We conclude that the risk of bill damage can potentially explainwhy oystercatchers avoid large bivalves and that oystercatchersmay maximize long-term intake rate by selecting prey sizes thatare "suboptimal" from a short-term rate-maximizing point ofview.