Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes.

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions. The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification. The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

The spread of incompatibility-inducing parasites in sub-divided host populations.

BACKGROUND:Maternally transmitted symbionts have evolved a variety of ways to promote their spread through host populations. One strategy is to hamper the reproduction of uninfected females by a mechanism called cytoplasmic incompatibility (CI). CI occurs in crosses between infected males and uninfected females and leads to partial to near-complete infertility. CI-infections are under positive frequency-dependent selection and require genetic drift to overcome the range of low frequencies where they are counter-selected. Given the importance of drift, population sub-division would be expected to facilitate the spread of CI. Nevertheless, a previous model concluded that variance in infection between competing groups of breeding individuals impedes the spread of CI.RESULTS:In this paper we derive a model on the spread of CI-infections in populations composed of demes linked by restricted migration. Our model shows that population sub-division facilitates the invasion of CI. While host philopatry (low migration) favours the spread of infection, deme size has a non-monotonous effect, with CI-invasion being most likely at intermediate deme size. Individual-based simulations confirm these predictions and show that high levels of local drift speed up invasion but prevent high levels of prevalence across the entire population. Additional simulations with sex-specific migration rates further show that low migration rates of both sexes are required to facilitate the spread of CI.CONCLUSION:Our analyses show that population structure facilitates the invasion of CI-infections. Since some level of sub-division is likely to occur in most natural populations, our results help to explain the high incidence of CI-infections across species of arthropods. Furthermore, our work has important implications for the use of CI-systems in order to genetically modify natural populations of disease vectors.

Presence of alternative lengthening of telomeres mechanism in patients with glioblastoma identifies a less aggressive tumor type with longer survival.

Patients with glioblastoma (GBM) have variable clinical courses, but the factors that underlie this heterogeneity are not understood. To determine whether the presence of the telomerase-independent alternative lengthening of telomeres (ALTs) mechanism is a significant prognostic factor for survival, we performed a retrospective analysis of 573 GBM patients. The presence of ALT was identified in paraffin sections using a combination of immunofluorescence for promyelocytic leukemia body and telomere fluorescence in situ hybridization. Alternative lengthening of telomere was present in 15% of the GBM patients. Patients with ALT had longer survival that was independent of age, surgery, and other treatments. Mutations in isocitrate dehydrogenase (IDH1mut) 1 frequently accompanied ALT, and in the presence of both molecular events, there was significantly longer overall survival. These data suggest that most ALT+ tumors may be less aggressive proneural GBMs, and the better prognosis may relate to the set of genetic changes associated with this tumor subtype. Despite improved overall survival of patients treated with the addition of chemotherapy to radiotherapy and surgery, ALT and chemotherapy independently provided a survival advantage, but these factors were not found to be additive. These results suggest a critical need for developing new therapies to target these specific GBM subtypes.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B.

Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. CONCLUSION: Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.

"Accomodating science" : a new way of thinking about rhetorical dynamics

By analyzing three case studies (neutrinos, victimization survey and quality of mass media), our present issue is to figure out if underlying successive accommodations to new rhetorical situations will have an impact on the respective importance of logos, ethos and pathos. We would like to pinpoint the stakes of science's public dimensions considering the scientists' image, their expertise, and also the given results' implication. We will especially take into account scientific papers that may be or are potentially controversial in the political, media and civic spheres.

HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy.

BACKGROUND: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements. METHODS: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART. RESULTS: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART. CONCLUSIONS: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

ELECTROCORTICAL DYNAMICS OF MOTOR INHIBITORY CONTROL WITH AGING

Motor inhibitory control plays a central role in adaptive behaviors during the entire lifespan. Inhibitory motor control refers to the ability to stop all (global) or a part (selective) of a planned or ongoing motor action. Although the neural processing underlying the global inhibitory control has received much attention from cognitive neuroscientists, brain modulations that occur during selective inhibitory motor control remain unknown. The aim of the present thesis is to investigate the spatio-temporal brain processes of selective inhibitory motor control in young and old adults using high-density electroencephalography. In the first part, we focus on early (preparatory period) spatio-temporal brain processes involved in selective and global inhibitory control in young (study I) and old adults (study II) using a modified Go/No-go task. In study I, we distinguished global from selective inhibition in the early attentional stage of inhibitory control and provided neurophysiological evidence in favor of the combination model. In study II, we showed an under-recruitment of neural resources associated with preservation of performance in old adults during selective inhibition, suggesting efficient cerebral and behavioral adaptations to environmental changes. In the second part, we investigate beta oscillations in the late (post-execution period) spatio-temporal brain processes of selective inhibition during a motor Switching task (i.e., tapping movement from bimanual to unimanual) in young (study III) and old adults (study IV). In study III, we identified concomitant beta synchronization related (i) to sensory reafference processes, which enabled the stabilization of the movement that was perturbed after switching, and (ii) to active inhibition processes that prevented movement of the stopping hand. In study IV, we demonstrated a larger beta synchronization in frontal and parietal regions in old adults compared to young adults, suggesting age-related brain modulations in active inhibition processes. Apart from contributing to a basic understanding of the electrocortical dynamics underlying inhibitory motor control, the findings of the present studies contribute to knowledge regarding the further establishment of specific trainings with aging. -- Le contrôle de l'inhibition motrice joue un rôle central dans les adaptations comportementales quel que soit l'âge. L'inhibition motrice se réfère à la capacité à arrêter entièrement (globale) ou en partie (sélective) une action motrice planifiée ou en cours. Bien que les processus neuronaux sous-jacents de l'inhibition globale aient suscité un grand intérêt auprès des neurosciences cognitives, les modulations cérébrales dans le contrôle de l'inhibition motrice sélective sont encore peu connues. Le but de cette thèse est d'étudier les processus cérébraux spatio-temporels du contrôle de l'inhibition motrice sélective chez les adultes jeunes et âgés en utilisant l'électroencéphalogramme à haute densité. Dans la première partie, nous comparons les processus cérébraux spatio-temporels précoces (préparation motrice) de l'inhibition sélective et globale chez des adultes jeunes (étude I) et âgés (étude II) en utilisant une tâche Go/No-go modifiée. Dans l'étude I, nous avons distingué l'inhibition globale et sélective au niveau des processus attentionnels précoces du contrôle de l'inhibition et nous avons apporté des preuves neurophysiologiques de l'existence d'un modèle de combinaison. Dans l'étude II, nous avons montré une sous-activation neuronale associée à un maintien de la performance dans l'inhibition sélective chez les adultes âgés, suggérant des adaptations cérébrales et comportementales aux contraintes environnementales. Dans la seconde partie, nous examinons les processus cérébraux spatio-temporels tardifs (post-exécution motrice) de l'inhibition sélective pendant une tâche de Switching (tapping bimanuel vers un tapping unimanuel) chez des adultes jeunes (étude III) et âgés (étude IV). Dans l'étude III, nous avons distingué des synchronisations beta liées (i) au traitement des réafférences sensorielles permettant de stabiliser le mouvement perturbé après le switching, et (ii) aux processus d'inhibition active afin d'empêcher les mouvements de la main arrêtée. Dans l'étude IV, cette synchronisation beta était plus forte dans les régions frontales et pariétales chez les âgés par rapport aux jeunes adultes suggérant des modulations cérébrales de l'inhibition active avec l'âge. Outre la contribution fondamentale sur la compréhension des dynamiques électrocorticales dans le contrôle de l'inhibition motrice, les résultats de ces études contribuent à développer les connaissances pour la mise en place de programmes d'entraînements adaptés aux personnes âgées.

Niche dynamics in space and time.

Niche conservatism, the tendency of a species niche to remain unchanged over time, is often assumed when discussing, explaining or predicting biogeographical patterns. Unfortunately, there has been no basis for predicting niche dynamics over relevant timescales, from tens to a few hundreds of years. The recent application of species distribution models (SDMs) and phylogenetic methods to analysis of niche characteristics has provided insight to niche dynamics. Niche shifts and conservatism have both occurred within the last 100 years, with recent speciation events, and deep within clades of species. There is increasing evidence that coordinated application of these methods can help to identify species which likely fulfill one key assumption in the predictive application of SDMs: an unchanging niche. This will improve confidence in SDM-based predictions of the impacts of climate change and species invasions on species distributions and biodiversity.

Effect of roscovitine on intracellular calcium dynamics: differential enantioselective responses.

Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.

Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism.

Background Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. Methods We exposed mice to the bacterium Escherichia coli and subsequently induced allergic airway inflammation through sensitization and intranasal challenge with ovalbumin. Results Pulmonary exposure to the bacterium Escherichia coli leads to a suppression of allergic airway inflammation. This immune modulation was neither mediated by the induction of a T helper 1 (Th1) response nor regulatory T cells; however, it was dependent on Toll-like receptor 4 (TLR4) but did not involve TLR desensitisation. Dendritic cell migration to the draining lymph nodes and activation of T cells was unaffected by prior exposure to E.coli, while dendritic cells in the lung displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production was abrogated. The suppression of airway hyper-responsiveness was mediated through the recruitment of gd T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of tumour necrosis factor a. Conclusion Our data reveal a localized immunoregulatory pathway that acts to protect the airways from allergic inflammation.

NO rebinding to myoglobin: a reactive molecular dynamics study.

The rebinding of NO to myoglobin after photolysis is studied using the 'reactive molecular dynamics' method. In this approach the energy of the system is evaluated on two potential energy surfaces that include the heme-ligand interactions which change between liganded and unliganded myoglobin. This makes it possible to take into account in a simple way, the high dimensionality of the transition seam connecting the reactant and product states. The dynamics of the dissociated NO molecules are examined, and the geometrical and energetic properties of the transition seam are studied. Analysis of the frequency of recrossing shows that the height of the effective rebinding barrier is dependent on the time after photodissociation. This effect is due mainly to protein relaxation and may contribute to the experimentally observed non-exponential rebinding rate of NO, as has been suggested previously.