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.

Every cigarette is doing your baby damage

This poster highlights the fact that smoking while pregnant increases the risk of low birth weight, premature birth, stillbirth and cot death, and directs women to the Smokers' Helpline.

Every cigarette is doing you damage

This poster was produced as part of the PHA's public information campaign on smoking.

Characterization of the gene expression related to the process of DNA damage tolerance in Schistosoma mansoni

In the course of its complex life cycle, the parasite Schistosoma mansoni need to adapt to distinct environments, and consequently is exposed to various DNA damaging agents. The Schistosoma genome sequencing initiative has uncovered sequences from genes and transcripts related to the process of DNA damage tolerance as the enzymes UBC13, MMS2, and RAD6. In the present work, we evaluate the importance of this process in different stages of the life cycle of this parasite. The importance is evidenced by expression and phylogenetic profiles, which show the conservation of this pathway from protozoa to mammalians on evolution.

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.

Patterns of recovery following focal hemispheric lesions : relationship between lasting deficit and damage to specialized networks

Rapport de synthèse : Objectif : Les déficits cognitifs présents dans la phase aiguë d'une lésion hémisphérique focale ont tendance à être de nature plus importante et plus générale que les déficits résiduels qui persistent dans la phase chronique de récupération. Nous avons investigué, dans le cadre de ce travail, les modèles de récupération auditive et la relation qui se dessine entre les déficits et les dommages relatifs à des réseaux spécifiques, pris comme modèle cognitif des fonctions auditives. De nombreuses études humaines dans les domaines de la neuropsychologie, de la psychophysique ainsi que des études d'activation suggèrent que les processus de reconnaissance et de localisation sonores sont effectués par l'intermédiaire de réseaux distincts tant sur le plan anatomique que fonctionnel : il s'agit des zones de traitement du «What» et du «Where », qui sont toutes deux présentes dans les deux hémisphères. Des études ont démontré que des lésions hémisphériques focales gauches ou droites, centrées sur ces réseaux, sont associées dans la phase chronique de récupération à des déficits correspondant en ce qui concerne la reconnaissance et/ou la localisation sonore. Méthode : Dans le cadre de ce travail, nous avons analysé les résultats concernant les performances auditives chez 24 patients ayant subi des lésions hémisphériques focales avec déficits secondaires dans des tâches de reconnaissance, de localisation et/ou de perception du mouvement sonore lors d'un premier testing effectué en phase aiguë (9 patients), en phase subaiguë (6 patients) ou en phase chronique précoce (9 patients). La totalité de ces patients ont bénéficié d'un second testing en phase chronique. Les observations effectuées ont servi à l'élaboration de patterns de récupération auditive. Résultats : Tous les 24 patients avaient initialement un déficit dans le domaine de la localisation et/ou de la perception du mouvement sonore. Dans la phase aiguë, ce déficit survenait sans atteinte spécifique du réseau «Where » chez presque la moitié des patients ; en revanche, cette situation n'était jamais observée chez les patients testés en phase chronique précoce. Une absence de récupération avait tendance à être associée à un dommage spécifique au réseau concerné ainsi qu'à la persistance d'un déficit au-delà de la phase aiguë. Les déficits résiduels n'étaient par ailleurs pas strictement en lien avec la taille lésionnelle ou l'étendue de l'atteinte du réseau spécifique. Conclusion : Nos résultats suggèrent que des mécanismes distincts sous-tendent la récupération et la plasticité à différentes périodes temporelles post-lésionnelles.

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-α blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

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.

Brain damage in methylmalonic aciduria: 2-methylcitrate induces cerebral ammonium accumulation and apoptosis in 3D organotypic brain cell cultures.

BACKGROUND: Methylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage. METHODS: To explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose-response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3. RESULTS: Among the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose-response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA. CONCLUSION: Surprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.

Peripheral inflammation increases the damage in animal models of nigrostriatal dopaminergic neurodegeneration: possible implication in Parkinson's disease incidence.

Inflammatory processes described in Parkinson’s disease (PD) and its animal models appear to be important in the progression of the pathogenesis, or even a triggering factor. Here we review that peripheral inflammation enhances the degeneration of the nigrostriatal dopaminergic system induced by different insults; different peripheral inflammations have been used, such as IL-1β and the ulcerative colitis model, as well as insults to the dopaminergic system such as 6-hydroxydopamine or lipopolysaccharide. In all cases, an increased loss of dopaminergic neurons was described; inflammation in the substantia nigra increased, displaying a great activation of microglia along with an increase in the production of cytokines such as IL-1β and TNF-α. Increased permeability or disruption of the BBB, with overexpression of the ICAM-1 adhesion molecule and infiltration of circulating monocytes into the substantia nigra, is also involved, since the depletion of circulating monocytes prevents the effects of peripheral inflammation. Data are reviewed in relation to epidemiological studies of PD.

Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients.

Background. DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. Conclusions. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

INTRODUCTION Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Remodeling of DNA replication structures by the branch point translocase FANCM

Fanconi anemia (FA) is a genetically heterogeneous chromosome instability syndrome associated with congenital abnormalities, bone marrow failure, and cancer predisposition. Eight FA proteins form a nuclear core complex, which promotes tolerance of DNA lesions in S phase, but the underlying mechanisms are still elusive. We reported recently that the FA core complex protein FANCM can translocate Holliday junctions. Here we show that FANCM promotes reversal of model replication forks via concerted displacement and annealing of the nascent and parental DNA strands. Fork reversal by FANCM also occurs when the lagging strand template is partially single-stranded and bound by RPA. The combined fork reversal and branch migration activities of FANCM lead to extensive regression of model replication forks. These observations provide evidence that FANCM can remodel replication fork structures and suggest a mechanism by which FANCM could promote DNA damage tolerance in S phase