Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines

L'exploration électrophysiologique de la face basale du lobe temporal et du lobe frontal : intérêt des électrodes sphénoïdales et intraorbitaires

National audienceThe authors have evaluated the interest of sphenoidal electrodes in detection of internal temporal spikes, and intra-orbital electrodes in the detection of orbito-frontal spikes. From a study of 26 patients, 21 with sphenoidal electrodes, 3 with intra-orbital electrodes and 2 with both electrodes, they observed the sensitivity and specificity of such electrodes in detecting spikes with no traduction upon extra-cranial electrodes, or with an unsuspected traduction as spikes at a distance from deep electrodes, or spikes on 2 foci, or bisynchronous discharges. Sphenoidal and intra-orbital electrodes constitute a non-invasive method that provides excellent information in the exploration of the mesiobasal cerebral face. Indications for the use of such a method are complex absences without EEG traduction or with an unsuspected traduction and without abnormalities on CT scan, in the context of functional surgery of epilepsy.Les auteurs ont évalué l'intérêt des électrodes extracrâniennes sphénoïdales et intraorbitaires dans l'exploration des pointes temporales internes, et des pointes orbitofrontales. À partir d'une étude sur 26 malades, 21 avec électrodes sphénoïdales, trois avec électrodes intraorbitaires et deux avec les deux types d'électrodes, ils démontrent la sensibilité et la spécificité de ces électrodes, capables de détecter des pointes n'ayant aucune traduction sur les électrodes de surface ou ayant une traduction en surface tout à fait inattendue comme des pointes en surface à distance de l'électrode implantée, ou sur deux foyers, ou même sous forme de décharges bilatérales et synchrones. Les électrodes sphénoïdales et intraorbitaires réalisent une méthode peu invasive qui apporte des renseignements inaccessibles par les autres électrodes extracrâniennes, dans l'exploration de la face mésiobasale de l'encéphale. Les indications de ces deux types d'électrodes qui nous semblent les plus judicieuses sont les crises partielles complexes dont l'expression électroencéphalographique classique est soit absente, soit imprécise

Relative luciferase activity in <i>TEP1</i> and <i>Lp</i> knockdown mosquitoes infected with <i>glyc::GFP-LUC</i>.

<p>Mosquitoes were injected with dsRNA prior to infection with <i>glyc::GFP-LUC</i>. Surviving mosquitoes 18–21 dpi were freeze dried, ground and the luciferase activity was measured. The values are normalised to the values in control treatment (<i>dsLacZ</i>). Shown are the results of 3 independent experiments for each treatment expressed as means of duplicate or triplicate measurements; error bars represent the standard error of the mean. The dotted line marks the value 1, corresponding to the respective controls. Sample sizes in experiment 1: <i>dsLacZ</i> (n = 21), <i>dsTEP1</i> (n<b> = </b>17); experiment 2: <i>dsLacZ</i> (n = 19), <i>dsTEP1</i> (n<b> = </b>17); experiment 3: <i>dsLacZ</i> (n = 26), <i>dsTEP1</i> (n<b> = </b>11); experiment 4: <i>dsLacZ</i> (n = 21), <i>dsLp</i> (n<i> = </i>20); experiment 5: <i>dsLacZ</i> (n = 26), <i>dsLp</i> (n = 8); experiment 6: <i>dsLacZ</i> (n = 44), <i>dsLp</i> (n = 39).</p

Generation of transgenic parasites expressing <i>gfp-luciferase</i> under salivary gland-specific promoters.

<p>(A) Schematic representation of a strategy used to obtain transgenic parasites. The top panel shows the wild type locus of <i>p230p</i>; arrows indicate the primers used to identify an intact <i>p230p</i> locus. The middle panel shows the linearised plasmid containing the 5′ and 3′ <i>p230p</i> fragments used for recombination (orange), <i>tgdhfr/ts</i> flanked by the 5′ UTR and 3′ UTR of <i>pbdhfr</i> (<i>tgdhfr/ts</i> cassette, yellow), the salivary gland-specific promoter (magenta), the <i>GFP-luciferase</i> fusion gene (green and blue) and a second <i>pbdhfr</i> 3′ UTR (dark blue). Crossing-over event is illustrated by the two crosses. The bottom panel shows the resulting disrupted, transgenic locus of <i>p230p</i>. Arrows represent the primers used for the identification of the disrupted <i>p230p</i> locus or of <i>luciferase</i>. Blue blocks illustrate the probes used for the FIGE analysis. (B) Southern analysis of FIGE separated chromosome of cloned transgenic lines confirms correct integration of the construct into the <i>230p</i> locus on chromosome 3. Hybridisation with the 3′UTR <i>dhfr/ts</i> probe recognises the integrated construct in chromosome 3 and the endogenous <i>P. berghei dhfr/ts</i> gene on chromosome 7. Note the more intense signal of chromosome 3 in the transgenic lines resulting from the two 3′UTR <i>dhfr/ts</i> regions in the construct. (C) Diagnostic PCR analyses of cloned transgenic parasites, confirming the correct integration of the constructs.</p

Correlation between sporozoite numbers and luciferase activity in whole mosquitoes, and dissected midgut and salivary gland sporozoites of <i>uis3::GFP-LUC</i> and <i>glyc::GFP-LUC</i>.

<p>Mosquitoes were infected with either <i>uis3::GFP-LUC</i> (A and C) or <i>glyc::GFP-LUC</i> (B and D) and sporozoites extracted from salivary glands (sgs), midguts (mgs) or whole mosquitoes were collected 18–19 dpi. The sporozoites and whole mosquitoes were lysed and dilution series of cell extracts were generated and used to perform luciferase assays. Luciferase activity was measured and plotted as the value after subtraction of the baseline against the sporozoite number (RLU). Goodness of the linear curve fit is given as r². Graphs representing two independent experiments are shown. 10⁴ sgs correspond to 1.1 mosquito equivalent in (C) and 1.2 in (D). Outliers have been omitted from the curve fit (i.e. highest sgs concentrations for isolated sgs for (C) and whole mosquitoes (D)).</p

GFP detection in blood and mosquito stages by fluorescence analysis.

<p>(A) For blood stage images, blood of infected mice was diluted and stained with the blue nuclear dye Hoechst 33258. Ookinetes were cultured, pelleted and stained with Hoechst 33258 before imaging. To image oocysts and salivary gland sporozoites, mosquitoes were infected with the transgenic parasites. Midguts containing oocysts were dissected directly into PBS/Hoechst 33258 11–21 dpi; infected salivary glands were dissected into PBS/Hoechst 33258 16–21 dpi. Oocysts and sporozoites were stained with the nuclear dye for 15–30 min. The GFP was visualized using the GFP fluorescence channel, the nuclei using the UV channel. The scale bars represent 5 µm for all the blood stages, ookinetes and sporozoites and 10 µm for oocysts.</p

Luciferase detection in mosquito stages by immuno-fluorescence assay (IFA).

<p>Luciferase expression in mgs and sgs was detected by immunofluorescence in dissected fixed midguts and salivary glands (17–21 days post infection) using anti-firefly luciferase antibody. Nuclei were visualized with DAPI. The scale bars represent 20 µm.</p

P09.084C - Strong interest of exome sequencing in progressive neurological diseases

International audienceIntroduction: Neurogenetics represents a vast, complex, ever changing discipline whose diagnosis currently remains challenging, since clinical and/or imaging features frequently appear very unspecific, especially early in the evolution (cerebellar ataxia, tremor, dystonia...). In molecular diagnosis, current strategies usually include sequential investigations that may lead to long, tedious, expensive and disappointing patients care. Exome sequencing (ES) appears a promising approach for neurogenetics, apart from when nucleotide motif expansion disorders can be suspected. Materials and Methods: We recruited 48 individuals without cognitive development impairment, referred to our center for suspected neurogenetic disease: 20 cerebellar ataxia (42%), 12 neuromuscular diseases (25%), 8 spastic paraplegia (17%), 2 abnormal movements (4%) and 6 others (12%) for whom the phenotype could not be labelled under a usual neurological syndrome. ES was interpreted in a solo-based strategy (94%) or in trio with parental pool (6%). Results: ES identified a causal diagnosis in 4/8 individuals with spastic paraplegia (50%), 3/6 “other” (50%), 1/2 with abnormal movements (50%), 5/12 with neuromuscular diseases (42%), 4/11 with isolated cerebellar ataxia (37%) and 2/9 with spinocerebellar ataxia (22%). Overall diagnostic yield was of 40 %. Conclusions: With such overall diagnostic yield, this study reinforces the diagnostic interest of ES in neurogenetics, in all its fields, as this diagnostic yield ranges from 22% in spinocerebellar ataxia (which is higher than current yield of gene panels) to 50% in spastic paraplegia. It also includes situations in which clinical displays may be complex and hard to systematize. First-tier implementation would significantly improve diagnostic yield in neurogenetics