Small-scale field evaluation of the efficacy and residual effect of Fludora® Fusion (mixture of clothianidin and deltamethrin) against susceptible and resistant Anopheles gambiae populations from Benin,West Africa

The emergence of mosquitoes that can avoid indoor-deployed interventions, such as treated bed nets and indoor residual spraying, threatens the mainstay of malaria control in Zambia. Furthermore, the requirement for high coverage of these tools poses operational challenges. Spatial repellents are being assessed to supplement these vector control tools, but limitations exist in the residual effect of the repellent and the need for external power or heat for diffusion of the volatiles. A semi-field evaluation of a novel controlled release spatial repellent device (CRD) was conducted in Macha, Zambia. These devices emanate metofluthrin with no need for external power. Devices were deployed in huts within the semi-field system (SFS). Female Anopheles gambiae sensu stricto released within the SFS were trapped overnight by light traps and collected by aspiration the next morning inside and outside of huts to determine the extent of mosquito repellency and the impact on host-seeking and survival. Experiments studied the impact of number of devices as well as the presence of hut occupants. The study was complemented with numerical methods based on computational fluid dynamics to simulate spatial distribution of metofluthrin. Presence of CRDs was associated with significant reductions in indoor counts of mosquitoes, regardless of whether huts were occupied or not. Repellency ranged from 15 to 60% compared to huts with no devices. Reducing the number of devices from 16 to 4 had little impact on repellency. When huts were occupied, indoor mosquito host-seeking was higher in the presence of CRDs, whilst survival was significantly reduced. This study demonstrated that deployment of as few as four CRDs within a hut was associated with reduced indoor mosquito densities. As would be expected, presence of occupants within huts, resulted in greater indoor catches (both with and without devices). The increased indoor mosquito host-seeking and mortality in huts when devices were present may be explained by the excito-repellency activity of metofluthrin. These semi-field experiments provide preliminary data on the utility of CRD spatial repellents to reduce indoor densities of An. gambiae mosquitoes. Studies will further investigate the impact of CRDs on mosquito behaviour as well as epidemiological protective efficacy.     

Two-Dimensional 1H NMR Study of a Tetradecapeptide with the Consensus Sequence Arg5-Asp-Val-Arg-Gly9: Structural Effects of the Outside Substitution Ser12 by Ala12

Abstract

Conformation of a tetradecapeptide with a RXVRG consensus sequence, Arg^s-Asp-Val-Arg-Gly⁹, found in several precursors of antibacterian peptides, was investigated in dimethylsulfoxide solution by proton NMR spectroscopy. Complete resonance assignments and conformational parameters were obtained through correlated (COSY) and nuclear Overhauser (NOESY) techniques. The ³J(αH, βH) coupling constants and the intramolecular NOE, NH…βH, were used to analyse the conformers around the Cα-Cβ bond and, in four cases, to obtain stereospecific assignments.

Use of restraints derived from NOE connectivities and ³J(NH, αH) coupling constants allows the determination of a range of φ and ψ dihedral angles for all the residues in the sequence. The present NMR results provide favourable evidence for the formation of two bends in the consensus sequence of the tetradecapeptide. The first one has most of the features of a Glu⁴- Val⁷ β–turn (low temperature coefficient of the Val⁷NH chemical shift, Arg⁵αH…Val⁷NH and Asp⁶NH.-.Val⁷NH NOE correlations). The second one exhibits only the Asp⁶αH…Arg⁷NH and Val⁷NH…Arg⁸NH NOE interactions. These consensus sequence organizations proposed were confirmed by molecular modeling based on low potential energy structure on the [4–9] fragment with high agreement of NOE data.

Overall, the substitution of Ser¹² by Ala¹² shifts the conformation of the hydrophobic moiety [10–14] towards a quite random coil structure in this fragment and strongly destabilizes the folded structures of the consensus domain where only one NH (Val⁷) is solvent-shielded opposed to three (Asp⁶ to Arg⁸) in the [Ser¹²] tetradecapeptide. These conformational changes could be related to the processing enzyme activities on these model oligopeptides.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

Influence of Cardiac Decentralization on Cardioprotection

The role of cardiac nerves on development of myocardial tissue injury after acute coronary occlusion remains controversial. We investigated whether acute cardiac decentralization (surgical) modulates coronary flow reserve and myocardial protection in preconditioned dogs subject to ischemia-reperfusion. Experiments were conducted on four groups of anesthetised, open-chest dogs (n = 32): 1- controls (CTR, intact cardiac nerves), 2- ischemic preconditioning (PC; 4 cycles of 5-min IR), 3- cardiac decentralization (CD) and 4- CD+PC; all dogs underwent 60-min coronary occlusion and 180-min reperfusion. Coronary blood flow and reactive hyperemic responses were assessed using a blood volume flow probe. Infarct size (tetrazolium staining) was related to anatomic area at risk and coronary collateral blood flow (microspheres) in the anatomic area at risk. Post-ischemic reactive hyperemia and repayment-to-debt ratio responses were significantly reduced for all experimental groups; however, arterial perfusion pressure was not affected. Infarct size was reduced in CD dogs (18.6±4.3; p = 0.001, data are mean±1SD) compared to 25.2±5.5% in CTR dogs and was less in PC dogs as expected (13.5±3.2 vs. 25.2±5.5%; p = 0.001); after acute CD, PC protection was conserved (11.6±3.4 vs. 18.6±4.3%; p = 0.02). In conclusion, our findings provide strong evidence that myocardial protection against ischemic injury can be preserved independent of extrinsic cardiac nerve inputs.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Self-association of adenine-dependent hairpin ribozymes

Hairpin ribozymes are flexible molecules that catalyse reversible self-cleavage after the docking of two independently folded internal loops, A and B. The activities, self-association and structures in solution of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) were studied by native gel electrophoresis, analytical centrifugation, and small angle neutron scattering. Bi-molecular RNA interactions such as linear–linear, loop–loop, loop–linear or kissing interactions have been found to be important in the control of various biological functions, and hairpin loops present rich potential for establishing both intra- and intermolecular interactions through standard Watson-Crick base pairing or non-canonical interactions. Similar results were obtained for ADHR1 and ADHR2. At room temperature, they indicated end-to-end self-association of the ribozymes in rod-like structures with a cross-section corresponding to two double strands side-by-side. Dimers, which predominate at low concentration (∼0.1 mg/ml), associate into longer rods, with increasing concentration (∼1 mg/ml). Above 65°C, the dimers and rods dissociated into compact monomers, with a radius of gyration similar to that of tRNA (about 70 bases). The dimers were non-active for catalysis, which suggests that dimer formation, probably by preventing the correct docking of loops A and B, could act as an inhibition mechanism for the regulation of hairpin ribozyme catalysis.