Laboratoire P.A.S.T.E.U.R

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Ultrafast, sensitive and large-volume on-chip real-time PCR for the molecular diagnosis of bacterial and viral infections

Lab Chip2016, Advance Article

 

To control future infectious disease outbreaks, like the 2014 Ebola epidemic, it is necessary to develop ultrafast molecular assays enabling rapid and sensitive diagnoses. To that end, several ultrafast real-time PCR systems have been previously developed, but they present issues that hinder their wide adoption, notably regarding their sensitivity and detection volume. An ultrafast, sensitive and large-volume real-time PCR system based on microfluidic thermalization is presented herein. The method is based on the circulation of pre-heated liquids in a microfluidic chip that thermalize the PCR chamber by diffusion and ultrafast flow switches. The system can achieve up to 30 real-time PCR cycles in around 2 minutes, which makes it the fastest PCR thermalization system for regular sample volume to the best of our knowledge. After biochemical optimization, anthrax and Ebola simulating agents could be detected in a 7-minute real-time PCR and a 7.5-minute reverse transcription real-time PCR (for 30 PCR cycles), respectively 6.4 and 7.2 times faster than with an off-the-shelf apparatus, while conserving real-time PCR sample volume, efficiency, selectivity and sensitivity. The highspeed thermalization also enabled us to perform sharp melting curve analyses in only 20s and to discriminate amplicons of different lengths by rapid real-time PCR. This real-time PCR microfluidic thermalization system is cost-effective, versatile and can be then further developed for point-of-care, multiplexed, ultrafast and highly sensitive molecular diagnoses of bacterial and viral diseases.

 

Inauguration de l'Institut Pierre-Gilles de Gennes (14 mars 2016)

L'Institut Pierre-Gilles de Gennes pour la Microfluidique, auquel prend part le département de Chimie, a été officiellement inauguré ce lundi 14 mars 2016 en présence de Mme Hidalgo (Maire de Paris), Mme Vallaud-Belkacem (Ministre de l'Enseignement et de la Recherche), M. Mandon (Secrétaire d'Etat chargé de l'ESR) et du Président de la République, M. François Hollande.

 

 
Carbon dioxide transport in molten calcium carbonate occurs through an oxo-Grotthuss mechanism via a pyrocarbonate anion

Nature Chemistry2016, Feb.

 

The reactivity, speciation and solvation structure of CO2 in carbonate melts are relevant both for the fate of carbon in deep geological formations and for its electroreduction to CO, to be used as fuel, by means of solvation in a molten carbonate electrolyte. In particular, the high solubility of CO2 in carbonate melts has been tentatively attributed to the formation of a new carbon species, the pyrocarbonate anion, C2O52-. In this work we study, by _rst principles molecular dynamics simulations, the behaviour of CO2 in molten calcium carbonate. We _nd that pyrocarbonate forms spontaneously and the identity of the CO2 molecule is quickly lost through O2- exchange.

The transport of CO2 in this molten carbonate thus occurs in a fashion similar to the Grotthuss mechanism in water, and is three times faster than molecular diffusion. This shows that Grotthuss- like transport is more general than thought so far.

Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

Scientific Reports 6, Article number: 19107 (2016)

 

Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity.

 

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Proceedings of the National Academy of Sciences, Volume 113 n°.3, January 2016, Pages 497-502

 

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a socalled fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.