Adsorption of titanium dioxide nanoparticles onto zebrafish eggs affects colonizing microbiota.

Teleost fish embryos are protected by two acellular membranes against particulate pollutants that are present in the water column. These membranes provide an effective barrier preventing particle uptake. In this study, we tested the hypothesis that the adsorption of antimicrobial titanium dioxide nanoparticles onto zebrafish eggs nevertheless harms the developing embryo by disturbing early microbial colonization. Zebrafish eggs were exposed during their first day of development to 2, 5 and 10 mg TiO2 L-1 (NM-105). Additionally, eggs were exposed to gold nanorods to assess the effectiveness of the eggs' membranes in preventing particle uptake, localizing these particles by way of two-photon microscopy. This confirmed that particles accumulate onto zebrafish eggs, without any detectable amounts of particles crossing the protective membranes. By way of particle-induced X-ray emission analysis, we inferred that the titanium dioxide particles could cover 25-45 % of the zebrafish egg surface, where the concentrations of sorbed titanium correlated positively with concentrations of potassium and correlated negatively with concentrations of silicon. A combination of imaging and culture-based microbial identification techniques revealed that the adsorbed particles exerted antimicrobial effects, but resulted in an overall increase of microbial abundance, without any change in heterotrophic microbial activity, as inferred based on carbon substrate utilization. This effect persisted upon hatching, since larvae from particle-exposed eggs still comprised higher microbial abundance than larvae that hatched from control eggs. Notably, pathogenic aeromonads tolerated the antimicrobial properties of the nanoparticles. Overall, our results show that the adsorption of suspended antimicrobial nanoparticles on aquatic eggs can have cascading effects across different life stages of oviparous animals. Our study furthermore suggests that aggregation dynamics may occur that could facilitate the dispersal of pathogenic bacteria through aquatic ecosystems.

The biodistribution and immuno-responses of differently shaped non-modified gold particles in zebrafish embryos.

Important questions raised in (nano)ecotoxicology are whether biodistribution of nanoparticles (NPs) is affected by particle shape and to what extent local adverse responses are subsequently initiated. For nanomedicine, these same questions become important when the labeled NPs lose the labeling. In this study, we investigated the biodistribution patterns of gold nanoparticles (AuNPs) as well as immune-related local and systemic sublethal markers of exposure and behavioral assessment. Hatched zebrafish embryos were exposed to four differently shaped non-coated AuNPs with comparable sizes: nanospheres, nanorods, nano-urchins, and nano-bipyramids. Shape-dependent trafficking of the particles resulted in a different distribution of the particles over the target organs. The differences across the distribution patterns indicate that the particles behave slightly different, although they eventually reach the same target organs - yet in different ratios. Mainly local induction of the immune system was observed, whereas systemic immune responses were not clearly visible. Macrophages were found to take AuNPs from the body fluid, be transferred into the veins and transported to digestive organs for clearance. No significant behavioral toxicological responses in zebrafish embryos were observed after exposure. The trafficking of the particles in the macrophages indicates that the particles are removed via the mononuclear phagocytic system. The different ratios in which the particles are distributed over the target organs indicate that the shape influences their behavior and eventually possibly the toxicity of the particles. The observed shape-dependent biodistribution patterns might be beneficial for shape-specific targeting in nanomedicine and stress the importance of incorporating shape-features in nanosafety assessment.

[Patients of a F-ACT youth team: juveniles with severe mental illness]. / Patiënten van een F-ACT-jeugdteam: jongeren met ernstige psychiatrische aandoeningen.

BACKGROUND: While three quarters of all psychiatric disorders develop before the age of eighteen, mental health services are barely enlisted by adolescents. This population is increasingly targeted by F-ACT youth teams (flexible assertive community treatment) in the Netherlands, considering regular mental health services often encounter difficulty in reaching out to them. It remains unknown how severe their psychopathology can be.
AIM: To describe the adolescents being treated in a F-ACT youth team, outlining their problems and needs. To answer the question whether youngsters under the age of eighteen can meet the criteria of severe mental illness (SMI).
METHOD: An inventory was made of all adolescents referred to our F-ACT youth team regarding psychiatric diagnosis and limitations in social functioning. Two case reports were included as subsequent illustration.
RESULTS: The juvenile population treated by our F-ACT youth team (n = 30) were predominantly males (n = 20). They suffered from various psychiatric problems, often coinciding with longstanding social limitations. Three quarters of these youths could meet the criteria of SMI.
CONCLUSION: It was established that a substantial amount of juveniles under the age of eighteen meet the criteria of SMI. This could facilitate the provision of targeted care for youths. However, there is also the risk of stigmatisation.

Revision of the Afrotropical Lycorininae (Ichneumonidae; Hymenoptera) II. Three new Lycorina species and additional distribution records.

Three new Afrotropical Lycorina species are described: L. horstmanni sp. nov., L. jacksonfive sp. nov. and L. riftensis sp. nov. The description of L. globiceps is expanded to include the large variability of the colour pattern. New distribution records are provided for L. fici and L. globiceps. An illustrated dichotomous key, and an online interactive matrix key available on www.waspweb.org, are provided for the identification of the seven known Afrotropical Lycorininae species.

Belesica madiba and Cremastus tutui (Ichneumonidae: Cremastinae), two entomological gems from South Africa.

The Afrotropical cremastine fauna (Hymenoptera: Ichneumonidae) is characterized by the near absence of the genus Cremastus, with a single species reported from Madagascar. The fauna is also characterized by the presence of several endemic genera. Among the latter is the exceptional monotypic genus Belesica. We describe two new species from these extremely rare Afrotropical genera, namely Belesica madiba sp. nov. and Cremastus tutui sp. nov.. Both are only known from South Africa.

Web-based participatory surveillance of infectious diseases: the Influenzanet participatory surveillance experience.

To overcome the limitations of the state-of-the-art influenza surveillance systems in Europe, we established in 2008 a European-wide consortium aimed at introducing an innovative information and communication technology approach for a web-based surveillance system across different European countries, called Influenzanet. The system, based on earlier efforts in The Netherlands and Portugal, works with the participation of the population in each country to collect real-time information on the distribution of influenza-like illness cases through web surveys administered to volunteers reporting their symptoms (or lack of symptoms) every week during the influenza season. Such a large European-wide web-based monitoring infrastructure is intended to rapidly identify public health emergencies, contribute to understanding global trends, inform data-driven forecast models to assess the impact on the population, optimize the allocation of resources, and help in devising mitigation and containment measures. In this article, we describe the scientific and technological issues faced during the development and deployment of a flexible and readily deployable web tool capable of coping with the requirements of different countries for data collection, during either a public health emergency or an ordinary influenza season. Even though the system is based on previous successful experience, the implementation in each new country represented a separate scientific challenge. Only after more than 5 years of development are the existing platforms based on a plug-and-play tool that can be promptly deployed in any country wishing to be part of the Influenzanet network, now composed of The Netherlands, Belgium, Portugal, Italy, the UK, France, Sweden, Spain, Ireland, and Denmark.

Floral volatiles, pollinator sharing and diversification in the fig-wasp mutualism: insights from Ficus natalensis, and its two wasp pollinators (South Africa).

Combining biogeographic, ecological, morphological, molecular and chemical data, we document departure from strict specialization in the fig-pollinating wasp mutualism. We show that the pollinating wasps Elisabethiella stuckenbergi and Elisabethiella socotrensis form a species complex of five lineages in East and Southern Africa. Up to two morphologically distinct lineages were found to co-occur locally in the southern African region. Wasps belonging to a single lineage were frequently the main regional pollinators of several Ficus species. In South Africa, two sister lineages, E. stuckenbergi and E. socotrensis, pollinate Ficus natalensis but only E. stuckenbergi also regularly pollinates Ficus burkei. The two wasp species co-occur in individual trees of F. natalensis throughout KwaZulu-Natal. Floral volatile blends emitted by F. natalensis in KwaZulu-Natal were similar to those emitted by F. burkei and different from those produced by other African Ficus species. The fig odour similarity suggests evolutionary convergence to attract particular wasp species. The observed pattern may result from selection for pollinator sharing among Ficus species. Such a process, with one wasp species regionally pollinating several hosts, but several wasp species pollinating a given Ficus species across its geographical range could play an important role in the evolutionary dynamics of the Ficus-pollinating wasp association.

Parasitoid fig-wasp evolutionary diversification and variation in ecological opportunity.

Ecological processes are manifest in the evolution and form of phenotype diversity. The great abundance of parasitoid species has led to speculation whether rates of speciation and extinction are dependent on parasitoid diversity. If these factors are mutually exclusive, species diversity should fluctuate instead of remaining relatively constant over time. It is not known whether radiations constrained by coevolutionary interactions conform to density-dependent diversification processes. Here we test the prediction that parasitoid fig wasp diversification responds to changes in ecological opportunity and density-independent processes. A phylogenetic approach is used to estimate relative divergence times and infer diversification rate changes using gamma-statistics. Monte Carlo constant rates tests that accommodate incomplete sampling could not reject constant rates diversification. Parasitoid fig wasp diversification is consistent with a more complex explanation than density-dependent cladogenesis. The results suggest contemporary African parasitoid fig wasp diversity remains a legacy of an ancient ecological opportunity facilitated by fig tree diversification following the breakup of Pan-African forests and evolution of the savanna biome over the last 55 Ma and the more recent aridification of the African continent in the last 5 Ma. These results imply that amplified phenotypic differentiation of specialist insects coevolving with plants is coupled to evolutionarily infrequent changes in ecological opportunity.

Internet-based monitoring of influenza-like illness in the general population: experience of five influenza seasons in The Netherlands.

Like in most other countries, influenza surveillance in The Netherlands is based upon influenza-like illness (ILI) consultations reported by sentinel general practitioners (GP). In addition, an internet-based monitoring of ILI in the general population started in 2003/2004 (Great Influenza Survey (GIS)). We compared GIS results over 5 influenza seasons with results from the GP system. Weekly ILI incidence from GIS correlated well with ILI incidence from the GP system the same week and even better 1 week later. This suggests that GIS is useful for early detection of trends in influenza activity. However, two important vulnerable groups, children and the elderly, are clearly underrepresented in the GIS. Furthermore, virological confirmation is lacking in the GIS. So, GIS can be a useful addition to the GP system, especially when representativeness can be improved and when participation remains at the current high level.

Gripenet: an internet-based system to monitor influenza-like illness uniformly across Europe.

Gripenet has been monitoring the activity of influenza-like-illness (ILI) with the aid of volunteers via the internet in the Netherlands and Belgium since 2003 and in Portugal since 2005. In contrast with the traditional system of sentinel networks of mainly primary care physicians coordinated by the European Influenza Surveillance Scheme (EISS), Gripenet obtains its data directly from the population. Any resident of the three countries can participate in Gripenet by completing an application form on the appropriate websites (http://www.griepmeting.nl for the Netherlands and Belgium, http://www.gripenet.pt for Portugal), which contains various medical, geographic and behavioural questions. Participants report weekly on the website any symptoms they have experienced since their last visit. ILI incidence is determined on the basis of a uniform case definition. In the 2006/2007 season, 19,623 persons participated in Gripenet in the Netherlands, 7,025 in Belgium and 3,118 in Portugal. The rise, peak and decline of ILI activity occurred at similar times according to Gripenet and EISS. However, ILI attack rates in the Netherlands (6.6%), Belgium (6.1%) and Portugal (5.6%) were remarkably more similar in Gripenet than in EISS (0.8%, 3.9%, and 0.6% respectively). Monitoring ILI activity with the direct participation of volunteers provides similar incidence curves compared to the traditional system coordinated by EISS. Whereas EISS provides an established system whose data is validated by virology tests, Gripenet is a fast and flexible monitoring system whose uniformity allows for direct comparison of ILI rates between countries. A current objective of Gripenet is to engage more European countries.

Patterns of diversification of Afrotropical Otiteselline fig wasps: phylogenetic study reveals a double radiation across host figs and conservatism of host association.

We studied the phylogenetic relationships of Otiteselline fig waSPS associated with Ficus in the Afrotropical region using rDNA sequences. African fig species usually host two species of Otiteselline fig waSPS. Phylogenetic analyses reveal that this pattern of association results from the radiation of two clades of waSPS superimposed on the fig system. Within each clade, wasp species generally cluster according to their host classification. The phylogenies of the two clades are also generally more congruent than expected by chance. Together these results suggest that Otiteselline wasp speciation is largely constrained by the diversification of their hosts. Finally, we show a difference in ovipositor length between the two Otiteselline species coexisting in the same Ficus species, which probably corresponds to ecological differences. The diversification of ecological niches within the fig is probably, with cospeciation, one of the key factors explaining the diversification and maintenance of species of parasites of the fig/pollinator system.

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion.

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.

High speed atomic force microscopy of biomolecules by image tracking.

An image-tracking procedure for atomic force microscopy is proposed and tested, which allows repeated imaging of the same area without suffering from lateral drift. The drift correction procedure is based on on-line cross-correlation of succeeding images. Using the image-tracking procedure allows zooming in on a small scan area over a long period and thus increases the frame rate inversely proportional to the scan area. Application of the procedure is demonstrated for diffusion of 5.4-kb DNA plasmids. With a scan area of 500 * 500 nm(2), a single plasmid can be imaged for more than 30 min at 4 s per frame, with a drift less than 10 nm. The high temporal resolution allows detailed analysis of the diffusion of DNA molecules. A diffusion coefficient of 30 nm(2)/s is found for most DNA molecules, though many molecules are temporally pinned to the mica surface, restricting diffusion.

Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscope.

Photolyase DNA interactions and the annealing of restriction fragment ends are directly visualized with the atomic force microscope (AFM). To be able to interact with proteins, DNA must be loosely bound to the surface. When MgCl2 is used to immobilize DNA to mica, DNA is attached to the surface at distinct sites. The pieces of DNA in between are free to move over the surface and are available for protein interaction. After implementation of a number of instrumental improvements, the molecules can be visualized routinely, under physiological conditions and with molecular resolution. Images are acquired reproducibly without visible damage for at least 30 min, at a scan rate of 2 x 2 microm2/min and a root mean square noise of less than 0.2 nm. Nonspecific photolyase DNA complexes were visualized, showing association, dissociation, and movement of photolyase over the DNA. The latter result suggests a sliding mechanism by which photolyase can scan DNA for damaged sites. The experiments illustrate the potential that AFM presents for modern molecular biology.