Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens

We have developed an efficient transformation system for the tropical actinorhizal tree Allocasuarina verticillata using Agrobacterium tumefaciens mediated gene transfer. Mature zygotic embryos were inoculated with the disarmed strain C58C1 carrying, in the binary vector BIN19, the nptll gene, providing kanamycin resistance as a selectable marker, and the reporter gene β-glucuronidase containing an intron. The transformed embryos were cultivated on nutrient medium supplemented with 0.5 µM NAA, 2.5 µM BA, 100 mg l⁻¹ kanamycin and 250 mg l⁻¹ cefotaxime. After 2 months, a 21% transformation frequency was obtained. Within 6–9 months, transgenic plants were recovered from 70% of the transformed calli. The presence of the transgenes was demonstrated by PCR analysis and by the expression of the β-glucuronidase; integration of the T-DNA was confirmed by Southern hybridization. More than 100 transgenic plants from a total of 23 independent transformation events have been successfully established in soil. The possibility to obtain nitrogen-fixing nodules after inoculation of transgenic A. verticillata plants by the actinomycetal strain of Frankia Allo2 was established.     

Introgression of the low-gossypol seed & high-gossypol plant trait in upland cotton: Analysis of [(Gossypium hirsutum?×?G. raimondii)2?×?G. sturtianum] trispecific hybrid and selected derivatives using mapped SSRs

In order to select genotypes of Gossypium hirsutum genetically balanced and expressing the low-gossypol seed & high-gossypol plant trait introgressed from the Australian wild diploid species G. sturtianum, the [(G. hirsutum × G. raimondii)2 × G. sturtianum] triple hybrid was backcrossed to G. hirsutum and autopollinated to produce backcross and selfed progenies. Two hundred and six mapped SSR markers of G. hirsutum were used to monitor the introgression of SSR alleles specific to G. sturtianum and G. raimondii in the selected progenies. A high level of heterozygosity, varying from 25 to 100%, was observed for all G. sturtianum-specific SSR markers conserved in the most advanced progenies. These results indicate the existence of segregation distortion factors that are associated with the genes controlling the researched trait. This study represents a starting point to map the genes involved in the expression of the trait and better understand its genetic determinism.     

A spatiotemporally coordinated cascade of protein kinase C activation controls isoform-selective translocation

In pituitary GH3B6 cells, signaling involving the protein kinase C (PKC) multigene family can self-organize into a spatiotemporally coordinated cascade of isoform activation. Indeed, thyrotropin-releasing hormone (TRH) receptor activation sequentially activated green fluorescent protein (GFP)-tagged or endogenous PKCbeta1, PKCalpha, PKCepsilon, and PKCdelta, resulting in their accumulation at the entire plasma membrane (PKCbeta and -delta) or selectively at the cell-cell contacts (PKCalpha and -epsilon). The duration of activation ranged from 20 s for PKCalpha to 20 min for PKCepsilon. PKCalpha and -epsilon selective localization was lost in the presence of G?6976, suggesting that accumulation at cell-cell contacts is dependent on the activity of a conventional PKC. Constitutively active, dominant-negative PKCs and small interfering RNAs showed that PKCalpha localization is controlled by PKCbeta1 activity and is calcium independent, while PKCepsilon localization is dependent on PKCalpha activity. PKCdelta was independent of the cascade linking PKCbeta1, -alpha, and -epsilon. Furthermore, PKCalpha, but not PKCepsilon, is involved in the TRH-induced beta-catenin relocation at cell-cell contacts, suggesting that PKCepsilon is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKCbeta1 activation, which in turn initiates a calcium-independent but PKCbeta1 activity-dependent sequential translocation of PKCalpha and -epsilon. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.     

Qualification of Standard Membrane-Feeding Assay with Plasmodium falciparum Malaria and Potential Improvements for Future Assays

Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA). The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds) to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability). We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in preclinical and early clinical development of transmission-blocking vaccines, and this study strongly supports its further development and application.     

Expression of Calcineurin Activity after Lung Transplantation: A 2-Year Follow-Up

The objective of this pharmacodynamic study was to longitudinally assess the activity of calcineurin during the first 2 years after lung transplantation. From March 2004 to October 2008, 107 patients were prospectively enrolled and their follow-up was performed until 2009. Calcineurin activity was measured in peripheral blood mononuclear cells. We report that calcineurin activity was linked to both acute and chronic rejection. An optimal activity for calcineurin with two thresholds was defined, and we found that the risk of rejection was higher when the enzyme activity was above the upper threshold of 102 pmol/mg/min or below the lower threshold of 12 pmol/mg/min. In addition, we report that the occurrence of malignancies and viral infections was significantly higher in patients displaying very low levels of calcineurin activity. Taken together, these findings suggest that the measurement of calcineurin activity may provide useful information for the management of the prevention therapy of patients receiving lung transplantation.     

Relationship between exposure to vector bites and antibody responses to mosquito salivary gland extracts

Mosquito-borne diseases are major health problems worldwide. Serological responses to mosquito saliva proteins may be useful in estimating individual exposure to bites from mosquitoes transmitting these diseases. However, the relationships between the levels of these IgG responses and mosquito density as well as IgG response specificity at the genus and/or species level need to be clarified prior to develop new immunological markers to assess human/vector contact. To this end, a kinetic study of antibody levels against several mosquito salivary gland extracts from southeastern French individuals living in three areas with distinct ecological environments and, by implication, distinct Aedes caspius mosquito densities were compared using ELISA. A positive association was observed between the average levels of IgG responses against Ae. caspius salivary gland extracts and spatial Ae. caspius densities. Additionally, the average level of IgG responses increased significantly during the peak exposure to Ae. caspius at each site and returned to baseline four months later, suggesting short-lived IgG responses. The species-specificity of IgG antibody responses was determined by testing antibody responses to salivary gland extracts from Cx. pipiens, a mosquito that is present at these three sites at different density levels, and from two other Aedes species not present in the study area (Ae. aegypti and Ae. albopictus). The IgG responses observed against these mosquito salivary gland extracts contrasted with those observed against Ae. caspius salivary gland extracts, supporting the existence of species-specific serological responses. By considering different populations and densities of mosquitoes linked to environmental factors, this study shows, for the first time, that specific IgG antibody responses against Ae. caspius salivary gland extracts may be related to the seasonal and geographical variations in Ae. caspius density. Characterisation of such immunological-markers may allow the evaluation of the effectiveness of vector-control strategies or estimation of the risk of vector-borne disease transmission.     

Estimating the burden of malaria in Senegal: Bayesian zero-inflated binomial geostatistical modeling of the MIS 2008 data

The Research Center for Human Development in Dakar (CRDH) with the technical assistance of ICF Macro and the National Malaria Control Programme (NMCP) conducted in 2008/2009 the Senegal Malaria Indicator Survey (SMIS), the first nationally representative household survey collecting parasitological data and malaria-related indicators. In this paper, we present spatially explicit parasitaemia risk estimates and number of infected children below 5 years. Geostatistical Zero-Inflated Binomial models (ZIB) were developed to take into account the large number of zero-prevalence survey locations (70%) in the data. Bayesian variable selection methods were incorporated within a geostatistical framework in order to choose the best set of environmental and climatic covariates associated with the parasitaemia risk. Model validation confirmed that the ZIB model had a better predictive ability than the standard Binomial analogue. Markov chain Monte Carlo (MCMC) methods were used for inference. Several insecticide treated nets (ITN) coverage indicators were calculated to assess the effectiveness of interventions. After adjusting for climatic and socio-economic factors, the presence of at least one ITN per every two household members and living in urban areas reduced the odds of parasitaemia by 86% and 81% respectively. Posterior estimates of the ORs related to the wealth index show a decreasing trend with the quintiles. Infection odds appear to be increasing with age. The population-adjusted prevalence ranges from 0.12% in Thillé-Boubacar to 13.1% in Dabo. Tambacounda has the highest population-adjusted predicted prevalence (8.08%) whereas the region with the highest estimated number of infected children under the age of 5 years is Kolda (13940). The contemporary map and estimates of malaria burden identify the priority areas for future control interventions and provide baseline information for monitoring and evaluation. Zero-Inflated formulations are more appropriate in modeling sparse geostatistical survey data, expected to arise more frequently as malaria research is focused on elimination.     

Phenotypic and genotypic characteristics of Acacia senegal (L.) Willd. root-nodulating bacteria isolated from soils in the dryland part of Senegal

Aims:  We characterized phenotypically and genotypically root-nodulating bacteria associated with Acacia senegal (L.) Willd. isolated from the soils surrounding A. senegal trees in the dry land area of Senegal.
Methods and Results:  The phenotypical and genotypical characterizations we carried out showed a high diversity of A. senegal root-nodulating bacteria. Phenotypic patterns showed adaptations of the rhizobial strains to many environmental stresses such as heat, drought, and salinity. Twelve molecular groups were distinguished by profiles obtained using polymerase chain reaction/restriction fragment length polymorphism techniques from intergenic spacer region rDNA. The highest genetic diversity was found around the A. senegal rhizosphere. Therefore, A. senegal seemed to have a positive influence on occurrence and genotypical diversity of rhizobial populations. Rhizobial isolates obtained in this study belonged phylogenetically to the genera Mesorhizobium and Rhizobium .
Conclusions:  Our results provided information about the genetic diversity of the rhizobial strains associated with A. senegal and suggested the adaptability of natural rhizobial populations to major ecological environmental stress within these soil environments.
Significance and Impact of the Study:  These results suggested a potential selection of compatible and well adapted strains under stress conditions as inoculants for successful A. senegal growth in arid lands.     

Experimental manipulation of termites (Isoptera,Macrotermitinae) foraging patterns in a Sahelo-Sudanese savanna: effect of litter quality

Summary The foraging pattern of four sympatric fungus-growing termite species was followed for five months of the dry season in a semi-arid savanna (Senegal). Experimental plots were established with and without termites (exclusion with insecticide) and amended with different litter quality: Acacia leaves, millet canes, ground millet (<500 µm), Combretum wood and cattle manure. Results showed different foraging patterns between the fungus-growing species. Odontotermes nilensis, for example, rapidly developed its foraging activity on the litter. In contrast, Macrotermes subhyalinus delayed its foraging activity but, extensively collected the food after its discovery. Differences in food preference were evident between the termite species: Odonto-termes nilensis foraged preferentially on millet, canes and Acacia leaves, Macrotermes subhyalinus preferred cane and ground millet. Ancistrotermes guineensis selected the millet, and Microtermes sp. was mostly recorded foraging on pieces of dead wood. Relationships between different foraging parameters were established to assess the relative intensity of this activity over the dry season. The comparison between untreated plots and plots treated with Fipronil® (Aventis) clearly demonstrated that the exclusion of termites from part of the savanna is possible and that this possibility can be used to study their role on the soil and organic matter cycle in situ.