A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome

<p>Abstract</p> <p>Background</p> <p>"Open" transcriptome analysis methods allow to study gene expression without <it>a priori </it>knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.</p> <p>Results</p> <p>In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.</p> <p>Conclusion</p> <p>We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.</p

La narcolepsie (approches transcriptomiques et physiopathologiques)

La narcolepsie se caractérise par des attaques de sommeil diurnes, des insomnies nocturnes, des crises de cataplexies (perte de tonus musculaire). Cette maladie serait due à une attaque autoimmune conduisant à la dégénérescence de neurones éveillants, les neurones à Hypocrétine ou Orexine. Quelle est la cible de l attaque autoimmune responsable de la narcolepsie ? Pour répondre à cette question, nous avons cherché à déterminer quels étaient les transcrits spécifiques des neurones à hypocrétine en comparant leur transcriptome à celui des neurones à MCH intacts chez les sujets narcoleptiques. Ainsi nous avons développé une méthode novatrice d analyse transcriptomique à haut débit. Les neurones à MCH sont des régulateurs du sommeil paradoxal, ont ils un rôle dans les manifestations anormales de ce sommeil chez les sujets narcoleptiques ? Nous avons réalisé une caractérisation neuroanatomique de ces cellules, puis nous avons évalué le taux de MCH dans le liquide céphalorachidien de sujets atteints d hypersomnieNarcolepsy is characterized by diurnal sleep attacks, nocturnal insomnia, cataplexy (loss of muscle tone). In this pathology, an autoimmune attack is said to be responsible for the loss of a wake active neuronal population, Hypocretin or Orexin neurons. What is the target of the autoimmune attack which is responsible for narcolepsy? We have searched for specific hypocretin transcripts by comparing hypocretin transcriptome to MCH transcriptome, a second neuronal population unaffected by narcolepsy. For this, we have developed an innovative approach of high flow transcriptomic analysis. MCH neurons are paradoxical sleep regulator : are these cells implicated in abnormal manifestations of this kind of sleep in narcoleptic patients ? We have carried out a neuroanatomical characterization of these cells, then we have evaluated the MCH levels in the cerebrospinal fluid of hypersomniac subjectsLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Could Sterile Aedes albopictus Male Releases Interfere with Aedes aegypti Population in Reunion Island?

International audienceIn Reunion Island, the feasibility of an Aedes albopictus control program using the Sterile Insect Technique (SIT) is studied. Because, in some regions, Ae. albopictus is living in sympatry with Aedes aegypti, the impact of releasing millions of sterile male Ae. albopictus on female Ae. aegypti reproduction needs to be assessed. Thus, to study the potential heterospecific matings, a marking technique using rhodamine B has been used. Rhodamine is given in solution to male mosquitoes to be incorporated into the male body and seminal fluid and transferred during mating into the bursa inseminalis and spermathecae of females. The presence of rhodamine in females occurred in 15% of cases when Ae. aegypti females were offered non-irradiated Ae. albopictus males, 5% when offered irradiated Ae. albopictus males and 18% of cases in the inverse heterospecific matings. Moreover, our results also showed that these matings gave few eggs but were not viable. Finally, the results showed that whatever the type of mating crosses, females in cages previously crossed with males of another species can re-mate with males of their species and produce an equivalent amount of egg compared to females only mated with conspecific males. Despite the promiscuity of the males and females in small cages for three days, heterospecific mating between sterile male Ae. albopictus and female Ae aegypti, 95% of the females have not been inseminated suggesting that in the field the frequency satyrization would be very low

Aedes albopictus Adult Medium Mass Rearing for SIT Program Development

International audienceFor the production of several hundred thousands of Aedes albopictus sterile males for the implementation of a Sterile Insect Technique (SIT) program, no costly mass-rearing equipment is needed during the initial phases, as optimized rearing at laboratory scale can be sufficient for the first steps. The aim of this study was to maximize the egg production by optimizing adult rearing methods for Ae. albopictus. The effect of parameters such as male/female ratio, density of adults, membrane type for blood feeding, quantity of blood delivered, continuous or discontinuous blood feeding, and surface of substrates for egg laying on overall egg production was tested to find optimized conditions. Based on the number of eggs produced per cage in response to the parameters tested, the optimum cage set-up was seen to be 1500 adults in a 30 × 30 × 30 cm cage with a male/female sex ratio of 1:3, fed by fresh bovine blood for periods of 30 min using a cellulose membrane covering a 10 cm stainless steel plate heated by a Hemotek device, and the provision of five oviposition cups to collect eggs. With this set-up, production per cage can reach a maximum of 35,000 eggs per week

Localization of the brainstem GABAergic neurons controlling paradoxical (REM) sleep

Paradoxical sleep (PS) is a state characterized by cortical activation, rapid eye movements and muscle atonia. Fifty years after its discovery, the neuronal network responsible for the genesis of PS has been only partially identified. We recently proposed that GABAergic neurons would have a pivotal role in that network. To localize these GABAergic neurons, we combined immunohistochemical detection of Fos with non-radioactive in situ hybridization of GAD67 mRNA (GABA synthesis enzyme) in control rats, rats deprived of PS for 72 h and rats allowed to recover after such deprivation. Here we show that GABAergic neurons gating PS (PS-off neurons) are principally located in the ventrolateral periaqueductal gray (vlPAG) and the dorsal part of the deep mesencephalic reticular nucleus immediately ventral to it (dDpMe). Furthermore, iontophoretic application of muscimol for 20 min in this area in head-restrained rats induced a strong and significant increase in PS quantities compared to saline. In addition, we found a large number of GABAergic PS-on neurons in the vlPAG/dDPMe region and the medullary reticular nuclei known to generate muscle atonia during PS. Finally, we showed that PS-on neurons triggering PS localized in the SLD are not GABAergic. Altogether, our results indicate that multiple populations of PS-on GABAergic neurons are distributed in the brainstem while only one population of PS-off GABAergic neurons localized in the vlPAG/dDpMe region exist. From these results, we propose a revised model for PS control in whic