The role of nicotinic acetylcholine receptors in lymphocyte development.

International audienceThe sizes of lymphocyte populations in lymphoid organs of nicotinic acetylcholine receptor knockout and chimera (knockout/wild-type) mice were studied by flow cytometry. The absence of beta2 subunit decreased, while nicotine treatment increased B lymphocyte numbers in the bone marrow. In chimera mice, either beta2 or alpha7 subunits influenced lymphocyte populations in primary lymphoid organs, while in the spleen, only alpha7 receptors were critical. More annexin V-positive B cells were found in the bone marrow of knockout than wild-type animals. We conclude that nicotinic receptors are involved in regulating lymphocyte development and control the B lymphocyte survival

Additional file 1: Supplementary figures 1 & 2. of FLIM-FRET analyzer: open source software for automation of lifetime-based FRET analysis

Figure S1. Comparison between FLIM-FRET analyzer and SPCImage software. Lifetime imaging measurement of the Cerulean Cyan Fluorescent Protein linked with Yellow Fluorescent Protein (CFP-YFP) chimera expressed in HEK-293 cells, using the microscope LEICA TCS SP2 combined with SPC-830 module (Becker & Hickl GmbH). A. Representative lifetime image was automatically segmented (red line) into four segmented cells which were independently analyzed by FLIM-FRET analyzer. B. Multi step process to analyze the fluorescence lifetime and distribution for each of the four cells using by SPCImage software. (The ROI in SPCImage software was manually selected.) C. The lifetime values calculated using FLIM-FRET analyzer shows high correlation (Pearson r > 0.99) with the values obtained with the SPCImage software. We additionally found the lifetime values of FLIM-FRET analyzer to be slightly longer than of the SPCImage, by a factor of 1.17±0.03. Figure S2. Validation of the FLIM-FRET analyzer using negative and positive FRET control probes expressed in cells. A. Lifetime images of CFP, CFP plus YFP, and CFP-YFP expressing HEK-293 cells processed with the FLIM-FRET analyzer. B. Comparative analysis of the fluorescence lifetime of single cells expressing CFP, CFP plus YFP, and CFP-YFP shows significant drop of the fluorescence lifetime for the C-Y chimera known to FRET. (PPTX 2044 kb

Additional file 2: of FLIM-FRET analyzer: open source software for automation of lifetime-based FRET analysis

User guide. (DOCX 4536 kb

Fluorogenic Quantum Dot-Gold Nanoparticle Assembly for Beta Secretase Inhibitor Screening in Live Cell

We have developed a novel fluorogenic nanoprobe prepared from the assembly of CdSe/ZnS quantum dot (QD) and gold (Au) nanoparticles in which QD was conjugated with a specifically designed β-secretase (BACE1) substrate peptide, which was allowed to bind to the Ni-nitrilotriacetate (Ni-NTA) modified Au nanoparticles. This coordination-mediated binding of the QD with Au nanoparticles via Ni-NTA-histidine (His) interaction resulted in highly efficient quenching of QD fluorescence through a distance-dependent fluorescence resonance energy transfer (FRET) phenomenon. The prequenched QD-Au assembly recovered the fluorescence in the presence of the BACE1 enzyme after incubation in vitro. The high quenching efficiency of AuNP and robust QD fluorescence signal recovery upon BACE1 enzymatic digestion enabled us to visualize BACE1 activity in living cells, which further allowed us to generate the half maximal inhibitory concentration (IC₅₀) values for BACE1 inhibitors in the cell-based assay utilizing a high throughput system (HTS). These results suggest the potential application of QD-AuNP assembly toward the HTS drug screening system as a robust and efficient probe to identify active molecules in BACE1-related diseases such as Alzheimer’s disease

Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions

International audienceRabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways

Kinome-Wide RNA Interference Screening Identifies Mitogen-Activated Protein Kinases and Phosphatidylinositol Metabolism as Key Factors for Rabies Virus Infection

International audienceThroughout the rabies virus (RABV) infectious cycle, host-virus interactions define its capacity to replicate, escape the immune response, and spread. As phosphorylation is a key regulatory mechanism involved in most cellular processes, kinases represent a target of choice to identify host factors required for viral replication. A kinase and phosphatase small interfering RNA (siRNA) high-content screening was performed on a fluorescent protein-recombinant field isolate (Tha RABV). We identified 57 high-confidence key host factors important for RABV replication with a readout set at 18 h postinfection and 73 with a readout set at 36 h postinfection, including 24 common factors at all stages of the infection. Amongst them, gene clusters of the most prominent pathways were determined. Up to 15 mitogen-activated protein kinases (MAPKs) and effectors, including MKK7 (associated with Jun N-terminal protein kinase [JNK] signalization) and DUSP5, as well as 17 phosphatidylinositol (PI)-related proteins, including PIP5K1C and MTM1, were found to be involved in the later stage of RABV infection. The importance of these pathways was further validated, as small molecules Ro 31-8820 and PD 198306 inhibited RABV replication in human neurons.IMPORTANCE Rabies virus relies on cellular machinery for its replication while simultaneously evading the host immune response. Despite their importance, little is known about the key host factors required for rabies virus infection. Here, we focused on the human kinome, at the core of many cellular pathways, to unveil a new understanding of the rabies virus infectious cycle and to discover new potential therapeutic targets in a small interfering RNA screening. The mitogen-activated protein kinase pathway and phosphatidylinositol metabolism were identified as prominent factors involved in rabies virus infection, and those findings were further confirmed in human neurons. While bringing a new insight into rabies virus biology, we also provide a new list of host factors involved in rabies virus infection