Immunological mechanism of action and clinical profile of disease-modifying treatments in multiple sclerosis.

Multiple sclerosis (MS) is a life-long, potentially debilitating disease of the central nervous system (CNS). MS is considered to be an immune-mediated disease, and the presence of autoreactive peripheral lymphocytes in CNS compartments is believed to be critical in the process of demyelination and tissue damage in MS. Although MS is not currently a curable disease, several disease-modifying therapies (DMTs) are now available, or are in development. These DMTs are all thought to primarily suppress autoimmune activity within the CNS. Each therapy has its own mechanism of action (MoA) and, as a consequence, each has a different efficacy and safety profile. Neurologists can now select therapies on a more individual, patient-tailored basis, with the aim of maximizing potential for long-term efficacy without interruptions in treatment. The MoA and clinical profile of MS therapies are important considerations when making that choice or when switching therapies due to suboptimal disease response. This article therefore reviews the known and putative immunological MoAs alongside a summary of the clinical profile of therapies approved for relapsing forms of MS, and those in late-stage development, based on published data from pivotal randomized, controlled trials.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

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.

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.

Role of Notch signalling in intestinal epithelium

Résumé: Chez les mammifères, les intestins sont les organes ayant le plus haut taux de renouvellement cellulaire dans l'organisme. L'épithélium intestinal se renouvelle complètement en moins d'une semaine. Il se compose de projections (villosités) et d'invaginations (cryptes) qui ont toutes deux des fonctions bien distinctes. Les cellules de l'intestin sont constamment produites à partir de cellules souches, situées dans la crypte, qui se différencient en cellules proliférantes transitoires, puis en cellules caliciformes, de Paneth, entéroendocrine ou en entérocytes. Ces cellules migrent dans leurs lieux spécifiques pour accomplir leur fonction physiologique pour finalement mourir. A cours de mon travail de thèse, j'ai étudié le rôle de la voie de signalisation de Notch dans le renouvellement cellulaire et dans le processus de l'homéostase des cellules de l'intestin marin en utilisant le système Cre-loxP pour induire la délétion des gènes Notch1, Notch2, Jaggedl et RBP-Jk. Bien que l'inactivation de Notch1 avec ou sans Jagged1, ou celle de Notch2, n'aboutissent à aucun phénotype, une déficience pour RBP-Jk, ou pour Notch1 et Notch2 simultanément, conduit au développement d'un impressionnant phénotype. Au niveau de la crypte, une rapide et importante modification des cellules apparaît: les cellules proliférantes sont devenues des cellules caliciformes qui ont perdu la capacité de se renouveler. Ces résultats impliquent la voie Notch en tant que nouvelle clé de voûte dans le maintien des cellules qui s'auto-renouvellent dans l'épithélium intestinal. Un rôle similaire a été proposé pour la voie Wnt, laquelle n'est cependant, pas affectée dans nos souris. C'est pourquoi ces deux voies sont essentielles dans le maintien de la prolifération dans les cryptes intestinales. Ce travail a aussi proposé un mécanisme par lequel la voie Notch contrôlerait l'intégrité du cycle cellulaire dans les cellules de la crypte intestinale, ceci en inhibant la transcription d'un inhibiteur du cycle cellulaire, la protéine p27KIP1. De plus, l'inactivation de RBP-Jk dans les adénomes développés par les souris APCmin induisent la différenciation de cellules tumorales en cellules caliciformes. Comme autre effet, la localisation histologique des cellules de Paneth est également affectée par la délétion de RBP-Jk ou de Notch1/Notch2, suggérant un rôle pour la voie Notch dans le compartiment des cellules de Paneth. Finalement, ce travail démontre que les cellules progénitrices de l'intestin ont besoin d'une convergence fonctionnelle des voie Wnt et Notch. Ces résultats préliminaires peuvent être considérés comme un concept pour l'utilisation d'inhibiteurs de secrétase-γ (inhibiteurs de Notch) à des fins thérapeutiques pour les cancers colorectaux. Summary The mammalian intestine has one of the highest cellular turnover rates in the body. The complete intestinal epithelium is renewed in less than a week. It is divided into spatially distinct compartments in the form of finger-like projections (villi) and flask-shaped invaginations (crypts) that are dedicated to specific functions. Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate and home to their specific compartments before dying after having fulfilled their physiological function. In this thesis project, the physiological role of the Notch signalling cascade in the marine intestine was studied. Inducible tissue specific inactivation of Notch1, Notch2, Jagged1 and RBP-Jk genes was applied to assess their role in the maintenance of intestinal homeostasis and cell fate determination. The analysis unequivocally revealed that Notch1, Notch1 and Jagged1 combined as well as Notch2 are dispensable for intestinal homeostasis and lineage differentiation. However, deficiency of RBP-Jk as well as the simultaneous inactivation of both Notch1 and Notch2 receptors unveiled a striking phenotype. In these mice, a rapid and massive conversion of proliferative crypt cells into post-mitotic goblet cells was observed. These results identify the Notch pathway as a key player for the maintenance of the proliferative crypt compartment. A similar role was implicated for the Wnt cascade, which, however, was not affected in the different tissue specific Notch signalling deficient mice. Thus, the Wnt and Notch signalling pathways are essential for the self-renewal capacity of the intestinal epithelium. Furthermore, our results suggest a molecular mechanism for Notch signalling mediated control of cell cycle regulation within the crypt. The Notch cascade inhibits expression of the cyclin-dependent kinase inhibitor p27KIP1 and thereby maintains proliferation of the intestinal progenitor cells. In addition, the inactivation of RBP-Jk in adenomas developed by APCmin mice resulted in the differentiation of tumour cells into goblet cells. Finally, Notch deficiency affected differentiated Paneth cells, suggesting that Notch may play a role in the Paneth cell compartment. In summary, this work clearly demonstrates that undifferentiated, proliferative cells in intestinal crypts require the concerted activation of the RBP-Jk-mediated Notch signalling and the Wnt cascade. In addition, our preliminary results can be considered as a "proof-of-principle" for the use of γ-secretase inhibitors for therapeutic modalities for colorectal cancer.

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.

Controlling cytokinesis in the fission yeast Schizosaccharomyces pombe : the role of dma1; and ubc8

ABSTRACT The fission yeast Schizosaccharomyces pombe is a single celled eukaryote that has proved to be an excellent model system for the study of cell cycle control. S. pombe cells are rod shaped and grow mainly by elongation at their tips. They divide by formation of medially-placed cell wall, or septum, which cleaves the cell in two. Once the cell commits itself to mitosis the site of division is determined by formation of an acto-myosin based contractile ring at the cell cortex. The ring is assembled in stages throughout mitosis and contracts at the end of anaphase, coincident with spindle disassembly. The contraction, but not the assembly, of the ring requires the signal transduction network called the septation initiation network or SIN. The core components of the SIN are three protein kinases (cdc7p, sidl p and sid2p) and their regulatory subunits (spg1 p, cdcl4p and moblp, respectively). Signalling is dependent upon the nucleotide status of the GTPase spgl p, which is regulated by a two-component GAP protein, cdc16p-byr4p. Signalling is thought to emanate from the spindle pole body, where core SIN components are anchored to a scaffold comprised of sid4p and cdc11p. Activation of the SIN requires the protein kinase plolp, which also has additional roles in mitosis. SIN signalling is tightly regulated to assure the proper co-ordination of mitosis and cytokinesis. Ectopic activation of the SIN in interphase can uncouple septum formation from mitosis, while deregulated SIN signalling leads to formation of cells with multiple septa that do not cleave. Regulators of SIN activity are therefore of considerable interest. This study has concentrated upon two of these, dma1 and ubc8. I have demonstrated that dmal becomes essential when SIN signalling is activated. This leads me to propose a tripartite model for regulation of the SIN during the mitotic cell cycle. Increased expression of dma1 inhibits SIN signalling and prevents cell division. To identify potential targets and mediators of this, multicopy suppressors of dma1 toxicity were identified. One of these, ubc8, is the subject of this thesis. Genetic and molecular analyses are consistent with the view that ubc8p acts as an inhibitor of the SIN Localisation of ubc8p indicates that it is a nuclear protein. The ubc8 gene is not essential, but in its absence cells are unable to prevent septum formation if progression through mitosis is impaired. These data suggest that it may be an effector of the spindle assembly checkpoint. Together, these data shed new light upon the mechanisms by which cytokinesis is regulated in S. pombe. RESUME La levure Schizosaccharomyces pombe est un eucaryote unicellulaire qui est un bon système d'étude du cycle cellulaire. Les cellules de S. pombe sont en forme de bâtonnets et poussent par allongement aux deux bouts. Elles se divisent en formant une paroi au milieu de la cellule, qui s'appelle un septum et qui sépare la cellule en deux. Une fois que la cellule est engagée dans la mitose, le site de clivage est déterminé par la formation d'un anneau contractile d'acto-myosine au niveau du cortex cellulaire. Cet anneau est séquentiellement assemblé au cours de la mitose et se contacte à la fin de l'anaphase, au moment où le fuseau mitotique et désassemblé. La contraction, mais non pas l'assemblage, de l'anneau dépend d'un réseau de signalisation appelé septation initiation netvvork' ou SIN. Les composants centraux du SIN sont trois kinases (cdc7, sidi et sid2) ainsi que leurs sous-unités régulatrices (spgl, cdc14 et mob1, respectivement). La signalisation dépend du nucléotide rattaché à la GTPase spgl qui est régulée par une GAP comprenant deux sous-unités cdc16 et byr4. La signalisation est présumée provenir du pôle du fuseau où les composants centraux du SIN sont ancrés grâce à un échafaudage comprenant sid4 et cdcl 1. La signalisation est étroitement régulée pour assurer une bonne coordination entre mitose et cytokinèse. Une activation ectopique du SIN en interphase peut découpler la formation du septum de la mitose, engendrant des cellules à multiples septa qui ne sont pas clivés. C'est pourquoi les régulateurs du SIN sont d'un intérêt considérable. Cette étude se concentre autour de deux ces régulateurs, dma1 et ubc8. J'ai montré que dma1 devient essentiel quand la signalisation du SIN est activée. Ceci m'amène à proposer un modèle en trois parties pour la régulation du SIN durant la mitose. Une expression élevée de dma1 inhibe la signalisation du SIN et empêche la division cellulaire. Afin d'identifier des substrats ou médiateurs potentiels de la toxicité de dma1, des supresseurs en copies multiples ont été identifiés. Un de ces supresseurs, ubc8, constitue le deuxième sujet de cette thèse. Les études génétiques et moléculaires suggèrent un rôle inhibiteur du SIN par ubc8. Ubc8p est une protéine nucléaire, non essentielle, mais en son absence les cellules ne peuvent pas restreindre la fomation du septum, lorsque la progression de la mitose est perturbée. Les données suggèrent que ubc8 pourrait être un effecteur de point de contrôle de l'assemblage du fuseau mitotique. Prises dans leur ensemble, ces données apportent un nouvel éclairage sur les mécanismes de régulation de la cytokinèse dans S. pombe.

Methicillin-resistant Staphylococcus aureus resensitization by protein synthesis inhibitors : proposal for a mechanism

RESUME Staphylococcus aureus est un important pathogène à gram-positif, à la fois responsable d'infections nosocomiales et communautaires. Le S. aureus résistant à la méthicilline est intrinsèquement résistant aux bêta-lactamines, inhibiteurs de la synthèse de la paroi bactérienne, grâce à une enzyme nouvellement acquise, la protéine liant la pénicilline 2A, caractérisée par une faible affinité pour ces agents et pouvant poursuivre la synthèse de la paroi, alors que les autres enzymes sont bloquées. Ce micro-organisme a également développé des résistances contre quasiment tous les antibiotiques couramment utilisés en clinique. Parallèlement au développement de molécules entièrement nouvelles, il peut être utile d'explorer d'éventuelles caractéristiques inattendues de médicaments déjà existants, par exemple en les combinant, dans l'espoir d'un potentiel effet synergique. Comprendre les mécanismes de tels effets synergiques pourrait contribuer à la justification de leur utilisation clinique potentielle. Récemment, un effet synergique contre le S. aureus résistant à la méthicilline a été décrit entre la streptogramine quinupristine-datfopristine et les bêta-lactamines, aussi bien in vitro qu'in vivo. Le présent travail a pour but de proposer un modèle pour le mécanisme de cette interaction positive et de l'étendre à d'autres classes d'antibiotiques. Premièrement, un certain nombre de méthodes microbiologiques ont permis de mieux cerner la nature de cette interaction, en montrant qu'elle agissait spécifiquement sur le S. aureus résistant à la méthicilline et qu'elle était restreinte à l'association entre inhibiteurs de la synthèse des protéines et bêta-lactamines. Deuxièmement, L'observation de l'influence des inhibiteurs de la synthèse des protéines sur la machinerie de la paroi bactérienne, c'est-à-dire sur l'expression des protéines liant la pénicilline, responsables de la synthèse du peptidoglycan, a montré une diminution de la quantité de ta protéine liant la pénicilline 2, connue pour posséder une activité de transglycosylation, indispensable au bon fonctionnement de la protéine liant la pénicilline 2A, responsable de la résistance à la méthicilline. Troisièmement, l'analyse fine de la composition du peptidoglycan extrait de bactéries, avant ou après traitement par des inhibiteurs de la synthèse des protéines, a montré des altérations corrélant avec leur capacité à agir en synergie avec les bêta-lactamines contre S. aureus résistant à ta méthicilline. Ces altérations dans les muropeptides pourraient représenter une signature de la diminution de la quantité de la protéine liant la pénicilline 2. Le modèle mécanistique retenu considère que les inhibiteurs de la synthèse des protéines pourraient diminuer l'expression de la protéine Liant la pénicilline 2, indispensable à la résistance à la méthiciltine, et que ce déséquilibre dans les enzymes synthétisant la paroi bactérienne pourrait générer une signature dans les muropeptides. SUMMARY Staphylococcus aureus is a major gram-positive pathogen causing both hospital-acquired and community-acquired infections. Methicillin- resistant Staphylococcus aureus is intrinsically resistant to the cell wall inhibitors beta-lactams by virtue of a newly acquired cell-wall-building enzyme, tow-affinity penicillin-binding protein 2A, which can build the wall when other penicillin-binding proteins are blocked. Moreover, the microorganism has developed resistance to virtually all non-experimental antibiotics. In addition of producing entirely new molecules, it is useful to explore unexpected features of existing drugs, for example by using them in combination, expecting drug synergisms. Understanding the mechanisms of such synergisms would help justify their putative clinical utilization. Recently, a synergism between the streptogramin quinupristin-dalfopristin and beta-lactams was reported against methicillin-resistant S. aureus, both in vitro and in vivo. The present work intends to propose a model for the mechanism of this positive interaction and to extend it to other drug classes. First, microbiological experimentation helped better defining the nature of this interaction, restricting it to methicillin-resistant S. aureus, and to the association of protein synthesis inhibitors with beta-lactams. Second, the observation of inhibitors of protein synthesis influence on the cell-wall-building machinery, i.e. on the expression of penicillin-binding proteins responsible for peptidoglycan synthesis, showed a decrease in the amount of penicillin-binding protein 2, known to provide a transglycosylase activity for glycan chain elongation, indispensable for the functionality of the low-affinity penicillin-binding protein 2A responsible for methicillin resistance. Third, the fine analysis of the peptidoglycan composition purified from bacteria before or after treatment with inhibitors of protein synthesis showed alterations that correlated with their ability to synergize with beta-lactams against methicillin-resistant S. aureus. These muropeptide alterations could be the signature of decrease in the amount of penicillin-binding protein 2. The retained mechanistic model is that inhibitors of protein synthesis could decrease the expression of penicillin-binding protein 2, wich is indispensable for methicillin-resistance, and that this imbalance in cell-wall-building enzymes could generate a muropeptide signature.

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.

An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells.

Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may co-reside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

Crystal structure of NLRC4 reveals its autoinhibition mechanism.

Nucleotide-binding and oligomerization domain-like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.

High-dose intravenous immunoglobulin treatment and cerebral vasospasm : a possible mechanism of ischemic encephalopathy?

A 46-year-old woman with a severe polyradiculoneuropathy treated with high-dose intravenous immunoglobulin (IVIg) presented an encephalopathy with increased blood flow velocities of the middle cerebral arteries (MCAs) detected by transcranial Doppler (TCD) studies. The similitude between this observation and another case recently reported of a patient suffering from Guillain-Barré syndrome (GBS) and cerebral blood flow abnormalities after IVIg treatment prompted us to investigate the responsibility of the IVIg therapy in the genesis of these blood flow alterations. We studied therefore by TCD 10 consecutive patients who underwent this treatment for different reasons. In 1 case we observed an asymptomatic, spontaneously reversible increase in the blood flow velocities of the MCAs consistent with a vasospasm and occurring 3-10 days after completion of the therapy. Stroke and ischemic encephalopathy have been reported as possible complications of IVIg treatment. In the case under discussion, clinical events appeared shortly after the administration of the IVIg therapy and responded favorably to a treatment with nimodipine. Other etiopathogenic mechanisms, in particular a CNS vasculopathic process related to the GBS itself, have to be considered as well. Further studies, with a larger number of patients, are therefore needed to evaluate the underlying mechanisms of blood flow abnormalities occurring sometimes in GBS patients after IVIg treatment.