NCube/papyrus huggingface dataset:
La connectivité structurelle de l'insula chez l'humain

The insula is a complex structure involved in a wide range of functions. Tracing studies on non-human primates reveal a wide array of cortical connections in the frontal (orbitofrontal and prefrontal cortices, cingulate areas, and supplementary motor area), parietal (primary and secondary somatosensory cortices) and temporal (temporal pole, auditory, prorhinal and entorhinal cortices) lobes. However, recent human tractography studies have not observed connections between the insula and the cingulate cortices, although these structures are thought to be functionally intimately connected. In this work, we try to unravel the structural connectivity between these regions and other known functionally connected structures, benefiting from a higher number of subjects and the latest state-of-the-art high angular resolution diffusion imaging (HARDI) tractography algorithms with anatomical priors. By performing a HARDI tractography analysis on 46 young normal adults, our study reveals a wide array of connections between the insula and the frontal, temporal, parietal and occipital lobes as well as limbic regions, with a rostro-caudal organization in line with tracing studies in macaques. Notably, we reveal for the first time in humans a clear structural connectivity between the insula and the cingulate, parahippocampal, supramarginal and angular gyri as well as the precuneus and occipital regions.

Keyphrases: Insula, Diffusion, Tractography, Connectivity, Insular
The biasing of the 5-HT4 receptor as an antidepressant target

Major Depression can be very damaging on the 8% of North Americans that will have it at least once within their lifetime. Current treatments of depression have been developed through the understanding of the monoamine hypothesis; whereby serotonin (5-HT) transporters or enzymes are blocked or inhibited in order to maintain the neurotransmitter in the synaptic cleft. Over a period of several weeks this increase in NT induces synaptic plasticity which increases the response sensitivity of 5-HT in the synaptic cleft. This form of treatment is effective on 40% of patients, while the remaining 60% have partial or no response. In mouse models of depression, changes in phosphorylation levels of CREB correlated to antidepressants (AD) effects. In the case of RS67333 AD effects appear within 2-3 days of treatment. The response can be evidenced as anti- anhedonic. RS67333 is specific agonist for 5-HT4 receptor, a G protein coupled receptor (GPCR) that is typically located post synaptically, in limbic regions. There are other ligands that are also specific to receptor 5-HT4: 5-HT, Zacopride, prucalopride and ML10302, but have not been observed to have the same anti-anhedonia effects of RS67333. How does RS67333 differ from the others in terms of signaling? We used bioluminescence resonance energy transfer (BRET) to study the interactions of 5-HT4R with the α β γ subunits of the G protein and we compared these associations with other known 5-HT4 agonists. We found that RS67333 was a partial agonist and activated a conformational change of the Gαs protein but did not activate a conformational change of Gαo protein. Prucaloride, 5-HT and Zacopride also induced a conformational change of the Gαs complex but not ML10302. For the formation of Gαo, Prucalorpide, ML10302 and 5-HT acted as agonists, but not Zacopride. We used an ELISA assay to measure ligand induced internalization to study receptor regulation. We found that RS67333 and ML10302 did not induce receptor internalization, while Zacopride, Prucalopride and 5-HT  were very effective. The suboptimal efficacy of current ADs stresses the need to develop drugs with other mechanisms of action. RS67333 has persistent anti-anhedonia effects after a short period of time and further studies of its signalling properties may open up a new generation of fast and long acting antidepressants.

Keyphrases: 5-HT, RS67333, BRET, AD, Antidepressant, CREB, Anhedonia, Zacopride, Prucalopride, ML10302, 5-HT4, Functional Selectivity, GPCR, 5-HT4 Receptor
Déterminants moléculaires du clivage protéolytique nécessaire à la fonction de la sous-unité CaVα2δ1 du canal calcique CaV1.2

Voltage-gated calcium channels CaV1.2 play an essential role in the regulation of cardiac excitability. Functional channels are formed by the CaVα1 subunit and the intracellular CaVβ and the extracellular CaVα2δ1 subunits. CaVα2δ1 are type I transmembrane proteins that undergo a posttranslational modification producing their association at the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. The molecular determinants required for the proteolytic cleavage of the recombinant CaVα2δ1 protein were studied using biochemical, immunocytochemical, fluorescence, and electrophysiological methods. Enzymatic treatment with a phospholipase C specific for the cleavage of phosphatidyl inositol lipids abolished the colocalisation of CaVα2δ1 with a plasma membrane marker as shown using live-cell confocal imaging. Single point mutations G1060I or G1061I in the predicted transmembrane CaVδ domain was shown to significantly reduce the cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging, and to prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2 currents. The isoleucine mutations were also associated with a change in the migration pattern of the C-terminal fragments suggesting that proteolytic processing was altered. Single glycine to alanine mutations preserved the protein properties but the double mutant G1060A/G1061A significantly impaired cell surface expression of CaVα2δ1 and its functional regulation of CaV1.2. Altogether our data support a model where one Glycine residue at position 1060 or 1061 is required to produce the dominant proteolytic cleavage of CaVα2δ1 and further suggest that the GPI-anchored form of CaVα2δ1 is essential for channel function.

Keyphrases: expression recombinante, calcium channels, cell surface density, GPI membrane anchor, recombinant expression, electrophysiology, flow cytometry, confocal imaging, membrane isolation
Risk of elevated intraocular pressure after ranibizumab injection in patients with neovascular age-related macular degeneration

Purpose: Conflicting evidence exists about the risk of chronic elevation of intraocular pressure (IOP) after ranibizumab injections for neovascular age-related macular degeneration. The goal of this study is to evaluate this risk. Methods: A retrospective cohort study of 161 people. Inclusion criteria included receiving at least three ranibizumab injections in one eye only and having at least 9 weeks of follow-up. Exclusion criteria included the presence of uncontrolled glaucoma or ocular hypertension at baseline (IOP>=21mmHg). Chronic IOP elevation was defined as an increase >5mmHg of IOP on at least 2 consecutive visits. Results: There was no difference in the percentage of injected eyes that experienced a chronic IOP increase (n=8, 5%) compared to the percentage of uninjected eyes that experienced an IOP increase (n=9, 6%). However, a greater number of anti-VEGF injections was associated with chronic IOP elevation (P=0.032). Other risk factors for chronic IOP elevation included diabetes, a lower baseline IOP, and a higher maximum IOP (P<0.05). Conclusions: A greater number of injections appears to increase the risk of chronic IOP elevation. Also, diabetics appear to be more at risk and may need more careful follow-up or preventive pharmacological treatment.

Keyphrases: ranibizumab, intraocular pressure, ranibizumab, age-related macular degeneration
À la recherche de meilleurs traitements analgésiques : interactions entre le récepteur opioïde δ et ses différents agonistes

Opioids are still nowadays the most efficacious pharmacological compounds available to treat the different types of pain, and therefore provide a therapeutic analgesia. However, repeated administration of those compounds lead to major secondary effects like respiratory depression, tolerance, but also it was shown that some opioid compounds could induce seizures. From a therapeutical point of view, there is a serious need for new and better analgesic treatments that do not elicit such adverse effects. Our lab has been studying for years the signaletics of G-protein coupled receptors like the opioid receptors, and their capacity for functional selectivity, especially more recently the one of the delta opioid receptor (DOP). Indeed, this receptor elicits fewer adverse effects compared to the mu opioid receptor (MOP) that is the main target of all clinically used opioids such as morphine. However, it seems like the DOP itself would be responsible for the pro-epileptic states previously described. Thus, despite initial promises of the delta agonists towards reducing adverse effects whilst providing analgesia, the pro-convulsive effects that some seem to elicit have induced a loss of interest towards the DOP, and so far none of its agonists have gone further than pre-clinical trials. However, it has been shown that not all of those DOP agonists had those pro-convulsive adverse effects. How to explain such a phenomenon? This is the question which the present research will be asking. Thus our goal is to obtain and compare pharmacological signatures of the agonists known for being pro-convulsive versus those that are not ; regarding the transduction of signals through the delta opioid receptor and its heterotrimeric G-Protein ; and also regarding one of its main effectors to induce analgesia, an inwardly rectifying potassium channel. This comparison will be done according to the classical parameters of pharmacology, such as efficacy and potency ; but also according to the newest operational model, with parameters such as affinity and transduction coefficients. In order to do so, bioluminescence resonance energy transfer or BRET, will be used in order to characterize and quantify the signalling pathways there implicated. This research is embedded in a vast collaboration context, in between laboratories around the world, and within those laboratories as well, in hope to be able to one day synthesize, better pharmacological compounds, capable of targeting only the pathways responsible for the desired effects, here analgesia ; without triggering the associated adverse effects, here pro-convulsive states. The culmination of this research could allow to impact the lives of millions of people throughout the world, and this is why it is more than important for us to keep on pursuing it.

Keyphrases: RCPG, Biais, Convulsions, G protein-coupled receptor, GPCR, Bioluminescence resonance energy transfer, BRET, Delta opioid receptor, DOP, Analgesia, Functional selectivity, Biased agonism, Seizures, Operational model
Natriuretic peptide receptor-C activation regulates vascular smooth muscle cell hypertrophy : molecular mechanisms

Hypertension is associated with vascular remodelling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMCs). We earlier showed the implication of enhanced expression of Gqα and PLCβ1 proteins in VSMCs from 16- week-old spontaneously hypertensive rats (SHR). The present study was undertaken to investigate whether C-ANP4-23, a natriuretic peptide receptor-C (NPR-C) agonist that has been shown to inhibit vasoactive peptide-induced enhanced protein synthesis in VSMCs, could attenuate VSMC hypertrophy in rat models of cardiac hypertrophy and to explore the underlying mechanisms contributing to this inhibition. For these studies, aortic VSMCs from 16-week-old SHR were used. The protein synthesis, a marker of hypertrophy, was determined by (3H)leucine incorporation and the expression of proteins was determined by Western blotting. Cell volume was determined by three-dimensional confocal imaging. The protein synthesis was significantly enhanced in VSMC from SHR as compared to WKY and C-ANP4-23 treatment attenuated the enhanced protein synthesis to WKY control levels. In addition, the enhanced expression of the AT1 receptor as well as Gqα and PLCβ1 proteins, enhanced levels of superoxide anion (O2 -), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as well as the increased expressions of NADPH oxidase 4 (Nox4) and p47phox exhibited by VSMC from SHR were all attenuated by C-ANP4-23 treatment. Furthermore, C-ANP4-23 also attenuated the enhanced activation of epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), insulin-like growth factor 1 receptor (IGF-1R) and the enhanced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), AKT and c-Src. These results indicate that C-ANP4-23, via the activation of NPR-C, attenuates VSMC hypertrophy through its ability to decrease the overexpression of the AT1 receptor and Gqα/PLCβ1 proteins, the enhanced oxidative stress, the increased activation of growth factors and the enhanced phosphorylation of the MAPK/AKT signalling pathway. Thus, it can be suggested that C-ANP4-23, an activator of NPR-C, may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis.

Keyphrases: Hypertension, NPR-C, AKT, MAPK, c-Src, SHR, VSMC, Oxidative stress, NO, Growth factor receptors, Gqα proteins
Molecular mode of action of Cry6Aa1, a new insecticidal Bacillus thuringiensis toxin

Cry6Aa1 is a new toxin produced by Bacillus thuringiensis (Bt), which displays insecticidal activity against the Western corn rootworm (WCRW). The present work demonstrates that Cry6Aa1 is a pore-forming toxin (PFT) in planar lipid bilayers (PLBs). Contrary to other Bt toxins tested so far, pore formation by Cry6Aa1 does not require protease pretreatment and takes place at doses that are two to three orders of magnitude lower than those required for other Bt toxins under similar conditions. Pore formation by Cry6Aa1 is pH-dependent; the conductances of the pores range between 31 and 689 pS under symmetrical 150 mM KCl conditions; they are cationic and display a complex kinetic behaviour. The treatment of the toxin with midgut juice (Cry6Aa1 WCR1) does not change the biophysical properties of the pores. However, the treatment with trypsin (Cry6Aa1 TT) affects their conductance and selectivity at pH 5.5 (the WCRW gut physiological pH). The incorporation in PLBs of native membrane material from WCRW midgut affects the behaviour of the Cry6Aa1 pores. The molecular determinants of the mode of action of this new PFT appear therefore to differ from those reported before for other Bt toxins. The three-dimensional (3-D) atomic structure of Cry6Aa1 has just been elucidated. It shows that the toxin assumes an α-helix-rich configuration, which is quite similar to that of the ClyA PFT produced by E. coli. Based on the data available for ClyA, we have studied how different changes in the N- and C-terminal regions of Cry6Aa1 affect its pore formation ability in PLBs.

Keyphrases: Bacillus thuringiensis, Western corn rootworm, Cry6Aa1, δ-endotoxine, Planar lipid bilayer, bacterial toxin, Bacillus thuringiensis, pore-forming toxin, Western corn rootworm
Stabilité et fonctionnalité des glycoprotéines de l’enveloppe du VIH-1 recombinant CRF01_AE : rôle de l’histidine en position 375

The envelope glycoproteins (Env) from human immunodeficiency virus type 1 (HIV-1) mediate viral entry. The binding of the HIV-1 gp120 glycoprotein to CD4 contributes to the attachment of the virus to the target cell and triggers conformational changes in gp120 that allow high-affinity binding to the chemokine receptor CCR5 ou CXCR4.  Contrary to all other Envs from the same phylogenetic group M, which possess a serine at position 375, the majority of CRF01_AE strains possess a histidine. This residue is part of the “Phe43” cavity, where residue 43 of CD4 engages with the gp120.  Here we evaluated the functional consequences of replacing this residue in two CRF01_AE Envs (CM244 and 92TH023) by a serine, present in all the other clades from group M. We observed that reversion of the 375 amino acid to a serine resulted in a loss of functionality of both CRF_AE Envs, as measured by a loss in infectivity and their ability to mediate cell-to-cell fusion. The observed phenotype was the result of a weak interaction with CD4, which was not due to defective processing or trimer stability of these Envs. Therefore, modification of the amino acid at position 375 has also altered the sensitivity of virus neutralization by sCD4, which was reduced. Importantly, mutation of certain residues of the gp120 inner domain layers were able to partially restore wild-type levels of infectivity and cell-to-cell fusion to both CRF01-AE H375S Envs, suggesting a co-evolution of the Phe43 cavity and the gp120 inner domain. An understanding of the differences between the CRF01_AE envelopes and envelopes from group M presenting a serine at position 375 will provide a better knowledge about the functionality of the envelope glycoproteins. Among other things, it would be possible to identify the mechanisms involved in conformational changes of glycoproteins as well as those involved in the escape of envelope recognition by Env specific antibodies. Moreover, these possible differences in CRF01_AE envelopes could be exploited for the development of different vaccine approaches.

Keyphrases: CRF01_AE, gp120, HIV-1, Envelope glycoproteins, phe43 cavity, Inner domain
Characterization of a novel class of anti-HCV agents targeting protein-protein interactions

Hepatitis C virus (HCV) is an important causative agent for liver diseases and is responsible for a worldwide pandemic affecting roughly 180 million individuals worldwide. Late diagnosis following the progression to fibrosis and to cirrhosis, in nearly 16% of chronic infections, is attributed to the absence of symptoms in the first years of infection. By exploiting membrane protein-protein interactions (PPI), live cell assays using bioluminescence resonance energy transfer (BRET) technology have previously been optimized to complete a comprehensive hepatitis C virus (HCV) protein interaction network. Using the groundwork laid by this network study, a high-throughput assay (HTS) cell-based assay was implemented to identify novel inhibitory compounds targeting an unreported NS3/4A-NS3/4A interaction. Approximately 110,000 compounds from a small-molecule collection were screened to monitor modulation of NS3/4A homodimerization and were discriminated based on specificity and potency. UM42811 was identified as a potential NS3/4A-NS3/4A interaction activator and found to have a promising antiviral activity boasting an excellent therapeutic window. Combined deep sequencing and mutation mapping have yielded a resistance profile based on statistical and functional probability pointing towards a novel inhibitory mechanism targeting the HCV NS3/4A independent from protease activity inhibition. Data from an HCV to host protein interaction network generated by our group was used to analyze alternative effects of UM42811 on interactions which potentially benefit viral persistence. NS3/4A-specific host interactors were heavily associated with nuclear and mitochondrial transport based on Gene Ontology (GO). Among these specific interactors, karyopherin subunit beta 1 (KPNB1) and heat shock protein 60 (HSP60) were selected for further study. Interestingly, co-immunoprecipitation experiments revealed that UM42811 was able to prevent both KPNB1-NS3/4A and HSP60-NS3/4A interactions. Moreover, functional and western analysis revealed the KPNB1-NS3/4A interaction to have deleterious effects on iv interferon stimulated gene (ISG) induction. Unexpectedly, analysis revealed a putative NS3/4A mediated cleavage of KPNB1. Overall, this study demonstrates the strength of cell-based PPI strategies in the identification of novel HCV antiviral compounds, characterizes a novel inhibitory mechanism for HCV and reveals a potentially novel viral immune evasion mechanism.

Keyphrases: ADD, BRET, Hepatitis C Virus, HCV, Direct-Acting Antivirals, DAA, High-Throughput Screening, HTS, Bioluminescence Resonance Energy Transfer, BRET, Protein-Protein Interaction, PPI, Resistance
Impact of ATP-dependent RNA Helicase DDX3X on Herpes Simplex Type 1 (HSV-1) Replication

siRNA screening of 49 host proteins that are known to be incorporated in the mature virions of herpes simplex virus type 1 (HSV-1) revealed the importance of at least 15 cellular proteins for viral infectivity (Stegen, C et al. 2013). Among these, was the human protein DDX3X, a DEAD-box ATP-dependent RNA helicase. This multifunctional protein participates in different stages of gene expression such as mRNA transcription, maturation, mRNA export and translation. DDX3X has been shown to be involved in the replication of several viruses such as human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) vaccinia virus (VACV) and hepatitis C virus (HCV). The exact role of DDX3X in HSV-1 replication cycle is not known. Here we sought to find the detailed interaction between DDX3X with HSV-1. Surprisingly, the down-regulation as well as overexpression of DDX3X, significantly reduced the infectivity of HSV-1, indicating that the virus is sensitive to the precise levels of DDX3X. Accordingly, when we rescued DDX3X back to its normal cellular levels by sequential transfection of DDX3X siRNA and siRNA resistant DDX3X plasmid, the virus was able to infect cells efficiently compare to wild-type conditions. Furthermore, the virus changes the localization of DDX3X and causes its aggregation at early times in the infection. However, the virus does not change the cellular levels of DDX3X in at least two of three different cell lines tested. Using a luciferase assay we were able to establish that this protein has no effect on the entry of HSV-1. In fact, depleting or overexpressing DDX3X impaired the production on newly assembled viral particles by blocking the expression of all classes of viral proteins at the transcription level. Despite the known role of DDX3X in the stimulation of innate immune response and interferon type I production, DDX3X appears to act on HSV-1 replication independently of this pathway. This highlights a novel route of action of DDX3X by acting at the transcription level and the consequent genome replication of a DNA virus. By better understanding such pathogen interactions, it might now be possible to design novel therapeutic approaches.

Keyphrases: DDX3X, Herpes simplex virus type 1, siRNA, Protein overexpression and depletion, Viral gene expression
Étude des effets de l’initiation précoce du traitement sur la réactivité immunitaire chez l’enfant infecté par le VIH-1

Several studies have shown early treated children are not able to develop a cell-mediated response [1]. However, the viral rebound after prolonged remission in the case of the "Mississippi baby" raises many questions about the ability of these children to develop a specific immune response despite HIV viral suppression in the long term [2, 3]. We currently have five cases with a similar profile to the "Mississippi baby" that were identified previously [4]. The objective of this project is to determine whether early treated children develop an immune cell-mediated response against HIV that is quantitatively and/or qualitatively different from that found in children treated later. This study showed that the magnitude and diversity of CTL responses of children treated early is lower than that observed in children treated later if possible.

Keyphrases: Lymphocytes T Cytotoxiques (LTC), HIV, Cytotoxic T lymphocytes (CTL), Early treatment, perinatal infection, long term viral suppression