Spatio-temporal optimization of sampling for bluetongue vectors (<em>Culicoides</em>) near grazing livestock

BACKGROUND: Estimating the abundance of Culicoides using light traps is influenced by a large variation in abundance in time and place. This study investigates the optimal trapping strategy to estimate the abundance or presence/absence of Culicoides on a field with grazing animals. We used 45 light traps to sample specimens from the Culicoides obsoletus species complex on a 14 hectare field during 16 nights in 2009. FINDINGS: The large number of traps and catch nights enabled us to simulate a series of samples consisting of different numbers of traps (1-15) on each night. We also varied the number of catch nights when simulating the sampling, and sampled with increasing minimum distances between traps. We used resampling to generate a distribution of different mean and median abundance in each sample. Finally, we used the hypergeometric distribution to estimate the probability of falsely detecting absence of vectors on the field. The variation in the estimated abundance decreased steeply when using up to six traps, and was less pronounced when using more traps, although no clear cutoff was found. CONCLUSIONS: Despite spatial clustering in vector abundance, we found no effect of increasing the distance between traps. We found that 18 traps were generally required to reach 90% probability of a true positive catch when sampling just one night. But when sampling over two nights the same probability level was obtained with just three traps per night. The results are useful for the design of vector monitoring programmes on fields with grazing animals

Spatial abundance and clustering of Culicoides (Diptera: Ceratopogonidae) on a local scale

BACKGROUND: Biting midges, Culicoides, of the Obsoletus group and the Pulicaris group have been involved in recent outbreaks of bluetongue virus and the former was also involved in the Schmallenberg virus outbreak in northern Europe. METHODS: For the first time, here we investigate the local abundance pattern of these two species groups in the field by intensive sampling with a grid of light traps on 16 catch nights. Neighboring trap catches can be spatially dependent on each other, hence we developed a conditional autoregressive (CAR) model framework to test a number of spatial and non-spatial covariates expected to affect Culicoides abundance. RESULTS: The distance to sheep penned in the corner of the study field significantly increased the abundance level up to 200 meters away from the sheep. Spatial clustering was found to be significant but could not be explained by any known factors, and cluster locations shifted between catch nights. No significant temporal autocorrelation was detected. CAR models for both species groups identified a significant positive impact of humidity and significant negative impacts of precipitation and wind turbulence. Temperature was also found to be significant with a peak at just below 16 degrees Celcius. Surprisingly, there was a significant positive impact of wind speed. The CAR model for the Pulicaris group also identified a significant attraction to the smaller groups of sheep placed in the field. Furthermore, a large number of spatial covariates which were incorrectly found to be significant in ordinary regression models were not significant in the CAR models. The 95% C.I. on the prediction estimates ranged from 20.4% to 304.8%, underlining the difficulties of predicting the abundance of Culicoides. CONCLUSIONS: We found that significant spatial clusters of Culicoides moved around in a dynamic pattern varying between catch nights. This conforms with the modeling but was not explained by any of the tested covariates. The mean abundance within these clusters was up to 11 times higher for the Obsoletus group and 4 times higher for the Pulicaris group compared to the rest of the field

The range of attraction for light traps catching Culicoides biting midges (Diptera: Ceratopogonidae)

BACKGROUND: Culicoides are vectors of e.g. bluetongue virus and Schmallenberg virus in northern Europe. Light trapping is an important tool for detecting the presence and quantifying the abundance of vectors in the field. Until now, few studies have investigated the range of attraction of light traps. METHODS: Here we test a previously described mathematical model (Model I) and two novel models for the attraction of vectors to light traps (Model II and III). In Model I, Culicoides fly to the nearest trap from within a fixed range of attraction. In Model II Culicoides fly towards areas with greater light intensity, and in Model III Culicoides evaluate light sources in the field of view and fly towards the strongest. Model II and III incorporated the directionally dependent light field created around light traps with fluorescent light tubes. All three models were fitted to light trap collections obtained from two novel experimental setups in the field where traps were placed in different configurations. RESULTS: Results showed that overlapping ranges of attraction of neighboring traps extended the shared range of attraction. Model I did not fit data from any of the experimental setups. Model II could only fit data from one of the setups, while Model III fitted data from both experimental setups. CONCLUSIONS: The model with the best fit, Model III, indicates that Culicoides continuously evaluate the light source direction and intensity. The maximum range of attraction of a single 4W CDC light trap was estimated to be approximately 15.25 meters. The attraction towards light traps is different from the attraction to host animals and thus light trap catches may not represent the vector species and numbers attracted to hosts

Effect of staurosporine on the mobility and invasiveness of lung adenocarcinoma A549 cells: an in vitro study

<p>Abstract</p> <p>Background</p> <p>Lung cancer is one of the most malignant tumors, representing a significant threat to human health. Lung cancer patients often exhibit tumor cell invasion and metastasis before diagnosis which often render current treatments ineffective. Here, we investigated the effect of staurosporine, a potent protein kinase C (PKC) inhibitor on the mobility and invasiveness of human lung adenocarcinoma A549 cells.</p> <p>Methods</p> <p>All experiments were conducted using human lung adenocarcinoma A549 cells that were either untreated or treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L staurosporine. Electron microscopy analyses were performed to study ultrastructural differences between untreated A549 cells and A549 cells treated with staurosporine. The effect of staurosporine on the mobility and invasiveness of A549 was tested using Transwell chambers. Western blot analyses were performed to study the effect of staurosporine on the levels of PKC-α, integrin β1, E-cadherin, and LnR. Changes in MMP-9 and uPA levels were identified by fluorescence microscopy.</p> <p>Results</p> <p>We demonstrated that treatment of A549 cells with staurosporine caused alterations in the cell shape and morphology. Untreated cells were primarily short spindle- and triangle-shaped in contrast to staurosporine treated cells which were retracted and round-shaped. The latter showed signs of apoptosis, including vacuole fragmentation, chromatin degeneration, and a decrease in the number of microvilli at the surface of the cells. The A549 cell adhesion, mobility, and invasiveness significantly decreased with higher staurosporine concentrations. E-cadherin, integrin β1, and LnR levels changed by a factor of 1.5, 0.74, and 0.73, respectively compared to untreated cells. In addition, the levels of MMP-9 and uPA decreased in cells treated with staurosporine.</p> <p>Conclusion</p> <p>In summary, this study demonstrates that staurosporine inhibits cell adhesion, mobility, and invasion of A549 cells. The staurosporine-mediated inhibition of PKC-α, induction of E-Cad expression, and decreased integrin β1, LnR, MMP-9, and uPA levels could all possibly contribute to this biological process. These results represent a significant step forward in the ongoing effort to understand the development of lung carcinoma and to design novel strategies to inhibit metastasis of the tumor by targeting the cell-adhesion, mobility and invasion of tumor cells.</p

Quantifying Dispersal of European Culicoides (Diptera: Ceratopogonidae) Vectors between Farms Using a Novel Mark-Release-Recapture Technique

Studying the dispersal of small flying insects such as Culicoides constitutes a great challenge due to huge population sizes and lack of a method to efficiently mark and objectively detect many specimens at a time. We here describe a novel mark-release-recapture method for Culicoides in the field using fluorescein isothiocyanate (FITC) as marking agent without anaesthesia. Using a plate scanner, this detection technique can be used to analyse thousands of individual Culicoides specimens per day at a reasonable cost. We marked and released an estimated 853 specimens of the Pulicaris group and 607 specimens of the Obsoletus group on a cattle farm in Denmark. An estimated 9,090 (8,918-9,260) Obsoletus group specimens and 14,272 (14,194-14,448) Pulicaris group specimens were captured in the surroundings and subsequently analysed. Two (0.3%) Obsoletus group specimens and 28 (4.6%) Pulicaris group specimens were recaptured. The two recaptured Obsoletus group specimens were caught at the release point on the night following release. Eight (29%) of the recaptured Pulicaris group specimens were caught at a pig farm 1,750 m upwind from the release point. Five of these were recaptured on the night following release and the three other were recaptured on the second night after release. This is the first time that movement of Culicoides vectors between farms in Europe has been directly quantified. The findings suggest an extensive and rapid exchange of disease vectors between farms. Rapid movement of vectors between neighboring farms may explain the the high rate of spatial spread of Schmallenberg and bluetongue virus (BTV) in northern Europe

Protein kinase Cepsilon is important for migration of neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility.</p> <p>Methods</p> <p>PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot.</p> <p>Results</p> <p>Stimulation with 12-<it>O</it>-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS.</p> <p>Conclusion</p> <p>PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration.</p

Cooperation between Engulfment Receptors: The Case of ABCA1 and MEGF10

The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway