QSAR modelling of synergists to increase the efficacy of deltamethrin against pyrethroid-resistant Aedes aegypti mosquitoes$.

Space spraying of deltamethrin allows the control of adult Aedes (Stegomyia) aegypti mosquitoes. Unfortunately, many vector control programs are threatened by the development of resistances that decrease the efficacy of this adulticide. Faced with this situation, we can either try to use another insecticide presenting a different mechanism of action or find a strategy that brings back the efficacy of the insecticide at a satisfying level to pursue its use in vector control. Restoration of the efficacy of an insecticide can be obtained by means of a synergist. In this context, QSAR modelling was used to find synergists to combine with deltamethrin for increasing its efficacy against resistant strains of Ae. aegypti. Seventy-four structurally diverse chemicals with their 24-hour LD50 values, obtained under the same experimental conditions on Ae. aegypti females, were used. Molecules were described by means of autocorrelation vectors encoding lipophilicity, molar refractivity, H-bonding acceptor and donor ability. A three-layer perceptron (TLP) was employed as statistical tool. The performances of the models were evaluated through the analysis of the prediction results obtained on the different training and test sets (80%/20%) as well as from an out-sample test set. A 6/4/1 TLP computed with the Broyden-Fletcher-Goldfarb-Shanno second-order training algorithm led to the best prediction results. The convergence was obtained in 132 cycles. The sum of squares was used as error function. The hidden and output activation functions were tanh and exponential, respectively. Various chemical structures were identified as potential synergists and searched for their commercial availability. Molecules of interest were tested in vivo on Ae. aegypti by using the susceptible reference Bora Bora strain and two resistant strains from Martinique island. This led to the identification of the PSM-05 molecule that shows interesting synergistic activity.

In silico models for predicting vector control chemicals targeting Aedes aegypti.

Human arboviral diseases have emerged or re-emerged in numerous countries worldwide due to a number of factors including the lack of progress in vaccine development, lack of drugs, insecticide resistance in mosquitoes, climate changes, societal behaviours, and economical constraints. Thus, Aedes aegypti is the main vector of the yellow fever and dengue fever flaviviruses and is also responsible for several recent outbreaks of the chikungunya alphavirus. As for the other mosquito species, the A. aegypti control relies heavily on the use of insecticides. However, because of increasing resistance to the different families of insecticides, reduction of Aedes populations is becoming increasingly difficult. Despite the unquestionable utility of insecticides in fighting mosquito populations, there are very few new insecticides developed and commercialized for vector control. This is because the high cost of the discovery of an insecticide is not counterbalanced by the 'low profitability' of the vector control market. Fortunately, the use of quantitative structure-activity relationship (QSAR) modelling allows the reduction of time and cost in the discovery of new chemical structures potentially active against mosquitoes. In this context, the goal of the present study was to review all the existing QSAR models on A. aegypti. The homology and pharmacophore models were also reviewed. Specific attention was paid to show the variety of targets investigated in Aedes in relation to the physiology and ecology of the mosquito as well as the diversity of the chemical structures which have been proposed, encompassing man-made and natural substances.

A multiplex PCR to differentiate the two sibling species of mosquitoes Ochlerotatus detritus and Oc. coluzzii and evidence for further genetic heterogeneity within the Detritus complex.

Internal transcribed spacer regions of ribosomal DNA were sequenced, and species-specific primers were designed to simplify the identification of two morphologically similar species of the Detritus complex, Ochlerotatus detritus and Ochlerotatuscoluzzii. Each newly designed primer was able to amplify a species-specific fragment with a different size. Samples from France and Germany were successfully tested. This new tool prompts for bio-ecological studies to refine basic knowledge on the bionomics of this species complex, towards a better control and prevention of ensuing nuisances. Moreover, ITS2 sequencing revealed the existence of (1) two distinct haplotypes of Oc. detritus that are sympatric and widely distributed along the French Atlantic and Mediterranean littorals and (2) a specific haplotype in mosquitoes sampled from Tunisia, raising the question of the taxonomic status of this North-African population.

The moth Hylesia metabus and French Guiana lepidopterism: centenary of a public health concern.

The females of the moths Hylesia metabus have their abdomens covered by urticating hairs looking like micro-arrows and causing a puriginous dermatitis to humans known as "papillonite" in French Guiana and also called yellowtail moth dermatitis or Caripito itch. The densities of the moths show great seasonal and annual variations depending on mechanisms mostly unknown. When H. metabus infestations occur, numerous cases of dermatologic manifestations are reported from people living near the mangrove swamps where the moths are developing. One hundred years after the first "papillonite" epidemic reported from French Guiana in 1912, the data presented herein summarize the actual state of knowledge on H. metabus biology and ecology and on the lepidopterism. Some recommendations are proposed for the surveillance and warning systems of H. metabus infestations and to avoid contact with the moths. Research priorities are suggested to improve the control against this problem emerging between nuisance and public health.

Influence of environmental factors on the response of a natural population of Daphnia magna (Crustacea: Cladocera) to spinosad and Bacillus thuringiensis israelensis in Mediterranean coastal wetlands.

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 microg L(-1)) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 microL L(-1)) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors.

Effects of spinosad and Bacillus thuringiensis israelensis on a natural population of Daphnia pulex in field microcosms.

Spinosad, a candidate biological larvicide for mosquito control, was evaluated for its effects on a field population of Daphnia pulex, using Bacillus thuringiensis serovar israelensis (Bti) as a reference larvicide. Microcosms (125L enclosures) were placed in a shallow temporary oligohaline marsh where D. pulex was present. Three concentrations of spinosad (8, 17 and 33 microg L(-1)) and two concentrations of Bti (0.16 and 0.50 microL L(-1)) were applied (5 replicates per concentration, including the controls). Effects of larvicides on D. pulex were evaluated after 2, 4, 7, 14 and 21d of exposure, through measurements of abundance and individual size. Dissipation of spinosad from the water phase was rapid. Four days after treatment, residue concentration represented 11.8%, 3.9% and 12.7% of the initial exposure level for the nominal concentrations of 8, 17 and 33 microg L(-1), respectively. Spinosyns A and D dissipated at similar rates. Analysis of abundance and size structure of the D. pulex population showed an impact of spinosad. Both survival and size structure were affected. However, at the lowest concentration (8 microg L(-1)), population recovered after the first week. In microcosms treated with Bti, the abundance of D. pulex was not affected but the size structure of the population changed after 21d. As compared to laboratory tests, the use of in situ microcosms improved the environmental risk assessment of larvicides, taking into account the influence of environmental factors (e.g., temperature, light, salinity) and intrinsic capacity of recovery of D. pulex under field conditions.

Response of fibroblast activity and polyelectrolyte multilayer films coating titanium.

OBJECTIVES: The study of surface properties is a recent and crucial issue in the biomaterial fields applied to Odontology. The reference biomaterial in dental implantology is titanium. The principal objective is a perfect bio-integration in the oral ecosystem, both in terms of mucosal and bone tissues. The aim of this work was to optimize the tissue-titanium interface by applying polyelectrolyte multilayer films on the surface of titanium. METHODS: The experimental study was undertaken on pure titanium samples. Two types of film ending with polycations or polyanions were selected. Both film types were built with a first poly(ethyleneimine) (PEI) base layer and composed either of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) or of hyaluronic acid (HA) and poly(l-lysine) (PLL) layers. Final architectures were as follows: PEI-(PSS/PAH)(10), or PEI-(PSS/PAH)(10)-PSS, or chemically cross-linked PEI-(HA/PLL)(10) or PEI-(HA/PLL)(10)-HA. An analysis of the physicochemical characteristics of the surfaces was carried out by tensiometry measurements (dynamic contact angle, wettability, contact angle hysteresis) and atomic force microscopy. A biological study with human fibroblasts was followed over a 7-day culture period at days 0, 2, 4 and 7 to observe the cellular response in terms of morphology (scanning electron microscopy) and viability (Mosmann's test). RESULTS: The results showed that polyelectrolyte multilayer films could be successfully deposited onto titanium as previously described for glass or composite. Fibroblast adhesion and proliferation was strongly dependent on film type. SEM observations of cells on the different films agreed with the viability cell test. Furthermore, films containing PSS/PAH generated a better cellular response than films containing cross-linked HA/PLL. CONCLUSION: PSS/PAH polyelectrolyte films coating titanium could represent a new approach for oral bio-integration with great potential for clinical application in the fields of dental implantology. More particularly, the specific biofunctionalization of PSS/PAH films coating titanium could be envisioned by introducing layers of molecules that encourage the bio-integration process between the films.

Effect of type of polymerization on different properties of dental composites.

The aim of this study is to assess the influence of plasma lamps on the properties of the composites compared to the influence of conventional polymerization. Vickers hardness tests, three-point bending tests, and measurement of the shrinkage marginal gap by scanning electron microscopy were carried out on three resin composites (Tetric Ceram, Z-100 and Inten-S) irradiated with to lamps (Flipo) plasma and Astralis 7 halogen lamps). With a 3-second exposure, the results of Vickers hardness and resistance to flexion (excepting values for Z-100) were lower for the composites cured by the Flipo plasma lamp, than after 40-second curing by the conventional halogen lamp (Astralis 7), notably at a depth of 3 mm. With a 5-second exposure the results of Vickers hardness and resistance to flexion obtained using the plasma lamp approached those obtained by using the halogen lamp. Whatever the polymerization protocol used, the measurements of the gap between the tooth and the filling are very similar except for Z-100/Astralis 7, for which shrinkage results are more important. For any one resin composite and lamp used, the shrinkage values obtained at a depth of 4 mm are twice higher than those obtained at the surface. In conclusion, for a 3-second exposure the level of polymerization obtained by plasma curing is lower than the one obtained by halogen curing, particularly in depth. On the other hand, 5-second plasma curing results recommends the use of this kind of lamp.

Mechanical properties of two orthodontic adhesive materials polymerized with halogen and plasma curing.

OBJECTIVES: The aim of this study is to compare the efficiency of two polymerization techniques (halogen curing--Astralis 7 and plasma curing--Flipo), with two orthodontic adhesive materials (Enlight, a composite resin, and Fuji Ortho LC, a glass ionomer cement). METHODS: The efficiency of the polymerization techniques was shown by two mechanical tests. The hardness test was carried out on the exposed and non-exposed surfaces using 10 x 4 x 3-mm samples, polymerized either by halogen curing (40 seconds) or by plasma curing (5 seconds). The three-point bending tests were carried out on 2 x 2 x 25-mm samples polymerized as above. The samples were kept 1 hr at room temperature, then for 24 hrs in distilled water at 37 degrees C. RESULTS: Whatever the polymerization technique used, the results are similar for hardness and flexion, with the exception of the hardness tests carried out after polymerization with the Flipo light on the surface not directly exposed. CONCLUSION: In orthodontic practice, both polymerization techniques can be used. But a multi-bracket session can be long, and the reduction of time spent in the chair obtained by using plasma lamps seems to make this technique preferable.

Residual activity of Bacillus thuringiensis serovars medellin and jegathesan on Culex pipiens and Aedes aegypti larvae.

Bacillus thuringiensis serovar medellin strain 163-131 and Bacillus thuringiensis serovar jegathesan (B.t.jeg.) strain 367 are very toxic to mosquito larvae. However, they are 10 times less toxic than Bacillus thuringiensis var. israelensis (B.t.i.) to mosquito larvae under laboratory conditions. Lyophilized powders were produced from these strains and their toxicities were compared to that of powder produced from the B.t.i. strain. Larvicidal activity was titrated using Aedes aegypti (Bora-Bora strain) larvae, with IPS82 powder as the standard. The efficacy of these powders in the field was determined using Culex pipiens (Montpellier strain) in Paris, France, and Ae. aegypti larvae (French Guiana strain) in Cayenne, French Guiana, in standardized conditions. Residual activity was also assessed in the laboratory, using Cx. pipiens (SLAB strain), in Montpellier, France. Any negative effect of direct sunlight, soil, or polluted water on the residual activity of the 3 powders was recorded. Increasing bacterial concentration by a factor of 8 had little effect on the duration of larvicidal activity, except in the presence of polluted water and when substrate was added. All powders had similar initial efficacies against both types of mosquito larvae, in all conditions except water rich in organic matter. Bacillus thuringiensis serovar medellin had the lowest residual activity, both in the laboratory and in the field, whereas B.t.jeg. remained toxic for as long as B.t.i.

Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay.

Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.

Cytokeratin expression in human oral gingival epithelial cells: in vitro regulation by titanium-based implant materials.

To evaluate whether cytokeratin expression in human oral epithelial cells could be influenced by implant materials used in dental surgery, passaged human oral gingival epithelial cells were seeded on commercially pure titanium (CP-Ti) or on Ti6Al4V titanium alloy. Confluence was achieved after about 15 days on both substrates. Cells formed at that time, an organized layer of densely packed polygonal cells, and harbored a filamentous cytokeratin network typical of epithelial cells. Immunochemistry and immunoblot analysis were used to detect modifications of the amount of individual CK polypeptides (CK7, 8, 13, 18 and 19) in function of the culture substrate. Results showed that the level of CK8, CK18 and CK19 expression was not altered whatever the culture substrate used. The expression of CK13 was reduced in epithelial cells cultured on the titanium alloy, as compared with commercially pure titanium. Conversely, the level of CK7 was higher on the Ti6Al4V alloy than on commercially pure titanium. This study suggests that titanium-based implant materials could influence differently the phenotype of oral gingival epithelial cells.

Enhancement of the endothelial production of prostacyclin by inhibitors of protein synthesis.

1. Pretreatment of bovine aortic endothelial cells with cycloheximide enhanced their capacity to release prostacyclin in response to adenosine 5'-triphosphate (ATP) and bradykinin. 2. The action of cycloheximide was time-dependent; it became detectable after a 1 h exposure to the cells and was maximal after 3 h. 3. Puromycin mimicked the effect of cycloheximide. For these two agents, the enhancement of prostacyclin release was obtained at concentrations producing a partial inhibition of protein synthesis. 4. Cycloheximide increased the mobilization of free arachidonic acid induced by ATP in bovine aortic endothelial cells. 5. In conclusion, the synthesis of new proteins is not involved in the stimulatory action of ATP and bradykinin on prostacyclin production by bovine aortic endothelial cells. Despite the short half-life of prostaglandin H synthase in endothelial cells, cycloheximide and puromycin enhanced the release of prostacyclin induced by agonists. Our data suggest that this release might be under the control of rapidly turning-over phospholipase inhibitory proteins.

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells.

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).

Effects of amiloride analogues on the production of prostacyclin by aortic endothelial cells.

1. The release of prostacyclin (PGI2) from bovine aortic endothelial cells stimulated by adenosine 5'-triphosphate (ATP) was decreased by amiloride analogues bearing alkyl groups on the 5-amino nitrogen atom, like 5-(N-ethyl-N-isopropyl)amiloride (EIPA), which are inhibitors of the Na+/H+ exchanger. Analogues substituted on a terminal guanidino nitrogen atom were not inhibitory. 2. The release of PGI2 induced by ATP was not significantly depressed in a Na+-poor medium or in a medium acidified to pH 6.9, two conditions known to inhibit the Na+/H+ exchanger. 3. Cytoplasmic alkalinization by ammonium chloride did not suppress the inhibitory action of EIPA. By itself, ammonium chloride decreased the response of endothelial cells to ionophore A23187 and ATP, whereas sodium acetate had no effect. 4. EIPA did not decrease the mobilization of free arachidonic acid induced by ATP. It inhibited the conversion of exogenous arachidonate into PGI2 and prostaglandin E2 (PGE2). 5. Although the intracellular pH was not measured in this study, it seems unlikely that cytoplasmic alkalinization via the activation of the Na+/H+ exchanger plays a significant role in the stimulatory action of ATP on the release of PGI2 from endothelial cells. The inhibition of that release by EIPA and other amiloride analogues might involve a direct effect on cyclo-oxygenase, although an action on the reacylation of free arachidonic acid cannot be excluded.

Stimulation of prostacyclin release from aortic smooth muscle cells by purine and pyrimidine nucleotides.

ATP and ATP gamma S(10-100 microM) stimulated the release of prostacyclin (PGI2) from bovine aortic smooth muscle cells. This effect was reproduced by UTP, ITP and partially by GTP. ADP and ADP beta S, the P2X-selective agonist alpha, beta-methylene ATP (APCPP), AMP and adenosine were all inactive. This effect of ATP gamma S was not inhibited by Reactive Blue 2, an antagonist of P2Y receptors. The stimulation of PGI2 production in aortic smooth muscle cells by these nucleotides thus seems to involve receptors distinct from both P2X and P2Y subtypes, which are responsible for smooth muscle contraction and PGI2 release from endothelial cells, respectively.