Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Le taux de détresse respiratoire du nouveau-né augmente, celui des césariennes aussi: et si ce n'était pas un hasard? [Respiratory distress of the neonate and the rate of caesarean section have increased over the last 30 years. Is there a link?]

Ces trente dernières années, on note en Suisse une augmentation significative de l'incidence du syndrome de détresse respiratoire (SDR) chez le nouveau-né (NN), touchant particulièrement les enfants avec un poids de naissance >2500 g. En même temps, le taux des césariennes (CS) s'est aussi accru. Une explication pour une éventuelle corrélation entre les deux évolutions est une augmentation en particulier des CS électives qui ont tendance à être planifiées à un terme précoce pour éviter la mise en travail spontanée. Suite à cela, le foetus est privé de différents mécanismes qui favorisent l'adaptation pulmonaire périnatale. Les bénéfices réels de la CS sur la morbidité tant foetale que maternelle ne doivent pas faire oublier que la CS est un facteur de risque pour le SDR du NN. Ce risque peut être diminué efficacement en planifiant une CS élective après 39 semaines révolues. In Switzerland, the rate of respiratory distress in neonates needing hospitalization has doubled over the last thirty years, concerning in particular babies weighing more than 2500 g. In the same time, the rate of Caesarean section (CS) has also multiplied. We suppose that a link between the two evolutions might be the increase of elective CS. They tend to be planned early at term to avoid the onset of spontaneous labour As a consequence, the foetus is deprived of different mechanisms helping pulmonary transition around birth. The potential benefits of CS regarding morbidity of foetus and mother should not overshadow that CS is a significant risk factor for respiratory problems of the neonate. This risk could be dramatically decreased by planning elective CS only after completed 39 weeks of gestation

Partial disjoint path for multi-layer protection in GMPLS networks

In this paper, different recovery methods applied at different network layers and time scales are used in order to enhance the network reliability. Each layer deploys its own fault management methods. However, current recovery methods are applied to only a specific layer. New protection schemes, based on the proposed partial disjoint path algorithm, are defined in order to avoid protection duplications in a multi-layer scenario. The new protection schemes also encompass shared segment backup computation and shared risk link group identification. A complete set of experiments proves the efficiency of the proposed methods in relation with previous ones, in terms of resources used to protect the network, the failure recovery time and the request rejection ratio

Enhanced multi-layer protection in multi-service GMPLS networks

This paper focuses on QoS routing with protection in an MPLS network over an optical layer. In this multi-layer scenario each layer deploys its own fault management methods. A partially protected optical layer is proposed and the rest of the network is protected at the MPLS layer. New protection schemes that avoid protection duplications are proposed. Moreover, this paper also introduces a new traffic classification based on the level of reliability. The failure impact is evaluated in terms of recovery time depending on the traffic class. The proposed schemes also include a novel variation of minimum interference routing and shared segment backup computation. A complete set of experiments proves that the proposed schemes are more efficient as compared to the previous ones, in terms of resources used to protect the network, failure impact and the request rejection ratio

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Exploring Cross-layer power management for PGAS applications on the SCC platform

Technological limitations and power constraints are resulting in high-performance parallel computing architectures that are based on large numbers of high-core-count processors. Commercially available processors are now at 8 and 16 cores and experimental platforms, such as the many-core Intel Single-chip Cloud Computer (SCC) platform, provide much higher core counts. These trends are presenting new sets of challenges to HPC applications including programming complexity and the need for extreme energy efficiency.In this work, we first investigate the power behavior of scientific PGAS application kernels on the SCC platform, and explore opportunities and challenges for power management within the PGAS framework. Results obtained via empirical evaluation of Unified Parallel C (UPC) applications on the SCC platform under different constraints, show that, for specific operations, the potential for energy savings in PGAS is large; and power/performance trade-offs can be effectively managed using a cross-layerapproach. We investigate cross-layer power management using PGAS language extensions and runtime mechanisms that manipulate power/performance tradeoffs. Specifically, we present the design, implementation and evaluation of such a middleware for application-aware cross-layer power management of UPC applications on the SCC platform. Finally, based on our observations, we provide a set of recommendations and insights that can be used to support similar power management for PGAS applications on other many-core platforms.

Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Intracortical connectivity of layer VI pyramidal neurons in the somatosensory cortex of normal and barrelless mice.

In mice, barrels in layer IV of the somatosensory cortex correspond to the columnar representations of whisker follicles. In barrelless (BRL) mice, barrels are absent, but functionally, a columnar organization persists. Previously we characterized the aberrant geometry of thalamic projection of BRL mice using axonal reconstructions of individual neurons. Here we proceeded with the analysis of the intracortical projections from layer VI pyramidal neurons, to assess their contribution to the columnar organization. From series of tangential sections we reconstructed the axon collaterals of individual layer VI pyramidal neurons in the C2 barrel column that were labelled with biocytin [controls from normal (NOR) strain, 19 cells; BRL strain, nine cells]. Using six morphological parameters in a cluster analysis, we showed that layer VI neurons in NOR mice are distributed into four clusters distinguished by the radial and tangential extent of their intracortical projections. These clusters correlated with the cortical or subcortical projection of the main axon. In BRL mice, neurons were distributed within the same four clusters, but their projections to the granular and supragranular layers were significantly smaller and their tangential projection was less columnar than in NOR mice. However, in both strains the intracortical projections had a preference for the appropriate barrel column (C2), indicating that layer VI pyramidal cells could participate in the functional columnar organization of the barrel cortex. Correlative light and electron microscopy analyses provided morphometric data on the intracortical synaptic boutons and synapses of layer VI pyramidal neurons and revealed that projections to layer IV preferentially target excitatory dendritic spines and shafts.

Une Suisse pas si paisible : la violence comme moyen de revendication politique (1950-2000)

L'objectif de cette recherche est d'interroger la place, la valeur et l'appréhension de la violence comme un moyen de revendication politique dans un pays comme la Suisse qui s'est construit un mythe de non-violence. Son système de démocratie directe, son aisance économique, sa stabilité politique et sa neutralité seraient censés l'épargner à la fois des insurrections violentes et des conflits internationaux. Toutefois, une recherche dans les archives de la presse nationale révèle que pour la période étudiée, entre 1950 et 2000, la Suisse a vécu les actions collectives violentes provenant des séparatistes et des anti-séparatistes jurassiens, de l'extrême gauche et de l'extrême droite. Le pays a également été le terrain des actions violentes provenant de mouvements de lutte armée clandestins allemands, italiens ainsi que des attentats organisés par des mouvements arabes, comme le Front Populaire de Libération de la Palestine (FPLP). Avec une approche mettant l'accent sur les acteurs, plusieurs axes de recherches ont été développés, notamment la fonction de l'action violente pour les individus et les mouvements qui y ont recouru afin d'exprimer une revendication, qu'elle ait été politique, sociale ou culturelle, et la façon dont le gouvernement suisse a perçu le danger représenté par les différents courants politiques. La question des réseaux développés avec les organisations de lutte armée clandestines a été abordée avec les cas de l'Allemagne et de l'Italie, pour ce qui est des mouvements établis en Europe, puis, avec les organisations issues des pays arabes, notamment le FPLP. L'analyse articule une réflexion à la fois empirique et théorique, ne perdant jamais de vue que la notion de violence est le lieu privilégié de jugements de valeur et que sa signification varie selon d'où provient le discours.

Oficina de vivência corporal: movimento, reflexão e apropriação de si mesmo

O presente artigo aborda uma atividade inovadora no campo de ensino na área de Enfermagem. Esta atividade, teórica - vivencial, que chamamos de "Oficina de vivência corporal", tem como propostas: promover a consciência corporal; a apreensão de fundamentos sobre corporalidade; e fomentar a reflexão sobre si e o corpo do "outro ", que é sujeito de cuidados em enfermagem. É desenvolvido com alunos do Primeiro ano do Curso de Graduação em Enfermagem - Universidade Federal de São Paulo - UNIFESPI EPM. Apresenta uma pesquisa descritiva, onde o fenômeno é descrito e explicado . Propõe-se investigar aspectos relativos a experiência de 202 alunos que participaram das atividades desenvolvidas nas oficinas, durante os anos de 1997, 1998 e 1999.