Surgical treatment of lamellar macular hole associated with epimacular membrane.

BACKGROUND:: Although the surgical treatment of full-thickness macular hole is well established, the utility of pars plana vitrectomy in the treatment of lamellar macular hole (LMH) remains less clear. The purpose of the study is to report functional results of surgical treatment of LMH associated with epiretinal membrane. METHODS:: Retrospective chart review of patients undergoing pars plana vitrectomy and peeling of epiretinal membrane and internal limiting membrane, with or without air or gas tamponade, for symptomatic LMH associated with epimacular membrane. RESULTS:: Forty-five eyes of 44 patients were operated for LMH associated with epimacular membrane between May 2000 and July 2009. Pars plana vitrectomy and membrane peeling were combined with air or gas tamponade in 43 of 45 cases. Mean logarithm of the minimum angle of resolution best-corrected visual acuity improved from 0.4 preoperatively to 0.13 postoperatively (P < 0.0001). Improvement in visual acuity ranged from 0 Early Treatment Diabetic Retinopathy Study (ETDRS) lines to 8.9 ETDRS lines (mean, 2.65 ETDRS lines). Visual acuity improved by ≥1 ETDRS line(s) in 40 of 45 eyes (89%) and by ≥2 ETDRS lines in 26 of 45 eyes (58%) after the surgical procedure. No patient lost vision. CONCLUSION:: This small retrospective study suggests that surgical treatment of LMH associated with epimacular membrane may improve visual acuity in symptomatic patients.

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

The glucose transporter 4 FQQI motif is necessary for Akt Substrate of 160-Kilodalton-dependent plasma membrane translocation but not Golgi-localized g-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment.

Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.

Report on a review of State employee grievance processes, settlement agreements entered into by the State and payments made during the period July 1, 2010 through June 30, 2014

Report on a review of State employee grievance processes, settlement agreements entered into by the State and payments made during the period July 1, 2010 through June 30, 2014

Mapping the Membrane Topology of Hepatitis C Virus Nonstructural Protein 4B

BACKGROUND: Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has only poorly been characterized to date. In particular, a precise membrane topology is thus far elusive. Here, we explored a novel strategy to map the membrane topology of HCV NS4B. METHODS: Selective permeabilization of the plasma membrane, maleimide-polyethyleneglycol (mPEG) labeling of natural or engineered cysteine residues and immunoblot analyses were combined to map the membrane topology of NS4B. Cysteine substitutions were introduced at carefully selected positions within NS4B and their impact on HCV RNA replication and infectious virus production analyzed in cell culture. RESULTS: We established a panel of viable HCV mutants with cysteine substitutions at strategic positions within NS4B. These mutants are infectious and replicate to high levels in cell culture. In parallel, we adapted and optimized the selective permeabilization and mPEG labeling techniques to Huh-7 human hepatocellular carcinoma cells which can support HCV infection and replication. CONCLUSIONS: The newly established experimental tools and techniques should allow us to refine the membrane topology of HCV NS4B in a physiological context. The expected results should enhance our understanding of the functional architecture of the HCV replication complex and may provide new opportunities for antiviral intervention in the future.

Emerging functions of myelin associated proteins during development neuronal plasticity and and neurpdegeneration.

Adult mammalian central nervous system (CNS) axons have a limited regrowth capacity following injury. Myelin-associated inhibitors (MAIs) limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo-A, MAG and OMgp, share two common neuronal receptors: NgR1, together with its co-receptors (p75(NTR), TROY and Lingo-1), and the recently described paired immunoglobulin-like receptor B (PirB). These proteins impair neuronal regeneration by limiting axonal sprouting. Some of the elements involved in the myelin inhibitory pathways may still be unknown, but the discovery that blocking both PirB and NgR1 activities leads to near-complete release from myelin inhibition, sheds light on one of the most competitive and intense fields of neuroregeneration study during in recent decades. In parallel with the identification and characterization of the roles and functions of these inhibitory molecules in axonal regeneration, data gathered in the field strongly suggest that most of these proteins have roles other than axonal growth inhibition. The discovery of a new group of interacting partners for myelin-associated receptors and ligands, as well as functional studies within or outside the CNS environment, highlights the potential new physiological roles for these proteins in processes such as development, neuronal homeostasis, plasticity and neurodegeneration.

Brain Spectrins 240/235 and 240/235E: Differential Expression During Development of Chicken Dorsal Root Ganglia in vivo and in vitro.

Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.

Membrane interaction of polymyxin B and synthetic analogues studied in biomimetic systems: implications for antibacterial action

Membrane-active antimicrobial peptides, such as polymyxin B (PxB), are currently in the spotlight as potential candidates toovercome bacterial resistance. We have designed synthetic analogs ofPxB in order to determine the structural requirements for membraneaction. Since the mechanism of action of PxB involves interaction withboth the outer membrane and the cytoplasmic membrane of Gramnegative bacteria, we have used an approach based on mimicking theouter layers of these membranes using monolayers, Langmuir-Blodgettfilms and unilamelar vesicles, and applying a battery of biophysicalmethods in order to dissect the different events of membraneinteraction. Collectively, results indicate that the PxB analogues act inthe bacterial membrane by the same mechanism than PxB, and that cationic amphipathicity determines peptide activity.

Selection processes in a citrus hybrid population using RAPD markers

The objective of this work was to evaluate the processes of selection in a citrus hybrid population using segregation analysis of RAPD markers. The segregation of 123 RAPD markers between 'Cravo' mandarin (Citrus reticulata Blanco) and 'Pêra' sweet orange (C. sinensis (L.) Osbeck) was analysed in a F1 progeny of 94 hybrids. Genetic composition, diversity, heterozygosity, differences in chromosomal structure and the presence of deleterious recessive genes are discussed based on the segregation ratios obtained. A high percentage of markers had a skeweness of the 1:1 expected segregation ratio in the F1 population. Many markers showed a 3:1 segregation ratio in both varieties and 1:3 in 'Pêra' sweet orange, probably due to directional selection processes. The distribution analysis of the frequencies of the segregant markers in a hybrid population is a simple method which allows a better understanding of the genetics of citrus group.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Photonic characteristics of Langmuir-Blodgett self-assembled monolayers of colloidal silica particles

Monodispersed colloidal crystals based on silica sub-micrometric particles were synthesized using the Stöber-Fink-Bohn process. The control of nucleation and coalescence result in improved characteristics such as high sphericity and very low size dispersion. The resulting silica particles show characteristics suitable for self-assembling across large areas of closely-packed 2D crystal monolayers by an accurate Langmuir-Blodgett deposition process on glass, fused silica and silicon substrates. Due to their special optical properties, colloidal films have potential applications in fields including photonics, electronics, electro-optics, medicine (detectors and sensors), membrane filters and surface devices. The deposited monolayers of silica particles were characterized by means of FESEM, AFM and optical transmittance measurements in order to analyze their specific properties and characteristics. We propose a theoretical calculation for the photonic band gaps in 2D systems using an extrapolation of the photonic behavior of the crystal from 3D to 2D. In this work we show that the methodology used and the conditions in self-assembly processes are decisive for producing high-quality two-dimensional colloidal crystals by the Langmuir-Blodgett technique.

H2O-δD-Fe-III relations of dehydrogenation and dehydration processes in magmatic amphiboles

Stable isotope compositions of a suite of magmatic amphiboles from alkaline basalts and andesitic rocks were examined to constrain the effects of degassing processes on the hydrogen isotope compositions. The Fe3+ (as Fe3+/Fe-total) and H2O contents, as well as the H isotope compositions of the amphiboles, differ markedly (27-58%, 0.5-2.2 wt%, -107 to -15 parts per thousand, respectively) but indicate systematic variations. The observed trends can be explained either as dehydrogenation or dehydration processes, both of which are coupled to oxidation processes, the latter most probably related to O2- substitution within amphiboles. The dehydrogenation-dehydration models can be used to assess the primary compositions of the magmas. As an important example, delta D values of amphiboles of Martian meteorites are discussed in a similar context. Copyright (c) 2006 John Wiley & Sons, Ltd.

External forcings, oceanographic processes and particle flux dynamics in Cap de Creus submarine canyon, NW Mediterranean Sea

Particle fluxes (including major components and grain size), and oceanographic parameters (near-bottom water temperature, current speed and suspended sediment concentration) were measured along the Cap de Creus submarine canyon in the Gulf of Lions (GoL; NW Mediterranean Sea) during two consecutive winter-spring periods (2009 2010 and 2010 2011). The comparison of data obtained with the measurements of meteorological and hydrological parameters (wind speed, turbulent heat flux, river discharge) have shown the important role of atmospheric forcings in transporting particulate matter through the submarine canyon and towards the deep sea. Indeed, atmospheric forcing during 2009 2010 and 2010 2011 winter months showed differences in both intensity and persistence that led to distinct oceanographic responses. Persistent dry northern winds caused strong heat losses (14.2 × 103 W m−2) in winter 2009 2010 that triggered a pronounced sea surface cooling compared to winter 2010 2011 (1.6 × 103 W m−2 lower). As a consequence, a large volume of dense shelf water formed in winter 2009 2010, which cascaded at high speed (up to ∼1 m s−1) down Cap de Creus Canyon as measured by a current-meter in the head of the canyon. The lower heat losses recorded in winter 2010 2011, together with an increased river discharge, resulted in lowered density waters over the shelf, thus preventing the formation and downslope transport of dense shelf water. High total mass fluxes (up to 84.9 g m−2 d−1) recorded in winter-spring 2009 2010 indicate that dense shelf water cascading resuspended and transported sediments at least down to the middle canyon. Sediment fluxes were lower (28.9 g m−2 d−1) under the quieter conditions of winter 2010 2011. The dominance of the lithogenic fraction in mass fluxes during the two winter-spring periods points to a resuspension origin for most of the particles transported down canyon. The variability in organic matter and opal contents relates to seasonally controlled inputs associated with the plankton spring bloom during March and April of both years.

Regulation of frizzled receptor activity at the plasma membrane : an interplay of the receptor, the trimeric G protein Go, and RGS protein and a scaffolding protein Kermit

G-protein-signaling pathways convey extracellular signals inside the cells and regulate distinct physiological responses. This type of signaling pathways consists of three major components: G-protein-coupled receptors (GPCRs), heterotrimeric G proteins (G-proteins) and downstream effectors. Upon ligand binding, GPCRs activate heterotrimeric G proteins to initiate the signaling cascade. Dysfunction of GPCR signaling correlates with numerous diseases such as diabetes, nervous and immune system deficiency, and cancer. As the signaling switcher, G-proteins (Gs, Gq/11, G12/13, and Gi/o) have been an appealing topic of research for decades. A heterotrimeric G-protein is composed of three subunits, the guanine nucleotide associated a-subunit, ß and y subunits. In general, the duration of signaling is determined by the lifetime of activated (GTP bound) Ga subunits. Identification of novel communication partners of Ga subunits appears to be an attractive way to understand the machinery of GPCR signaling. In our lab, we mainly focus on Gao, which is abundantly expressed in the nervous system. Here we present two novel interacting partners of Drosophila Gao: Dhit and Kermit, identified through yeast two-hybrid screening and genetic screening respectively. Dhit is characterized by a small size with a conserved RGS domain and an N-terminal cysteine rich motif. The RGS domain possesses the GAP (GTPase activating protein) activity towards G proteins. However, we found that Dhit exerts not only the GAP activity but also the GDI (guanine nucleotide dissociation inhibitor) activity towards Gao. The unexpected GDI activity is preserved in GAIP/RGS19 - a mammalian homologue of Dhit. Further experiments confirmed the GDI activity of Dhit and GAIP/RGS19 in Drosophila and mammalian cell models. Therefore, we propose that Dhit and its mammalian homologues modulate GPCR signaling by a double suppression of Ga subunits - suppression of their nucleotide exchange with GTP and acceleration of their hydrolysis of GTP. Kermit/GEPC was first identified as a binding partner of GAIP/RGS19 in a yeast two- hybrid screen. Instead of interacting with the Drosophila homologue of GAIP/RGS19 (Dhit), Kermit binds to Gao in vivo and in vitro. The functional consequence of Kermit/Gao interaction is the regulation of localization of Vang (one of the planar cell polarity core components) at the apical membrane. Overall, my work elaborated the action of Gao with its two interaction partners in Gao- mediated signaling pathway. Conceivably, the understanding of GPCR signaling including Gao and its regulators or effectors will ultimately shed light on future pharmaceutical research. - Les voies de signalisation médiées par les protéines G transmettent des signaux extracellulaires à l'intérieur des cellules pour réguler des réponses physiologiques distinctes. Cette voie de signalisation consiste en trois composants majeurs : les récepteurs couplés aux protéines G (GPCRs), les protéines G hétérotrimériques (G-proteins) et les effecteurs en aval. Suite à la liaison du ligand, les GPCRs activent les protéines G hétérotrimériques qui initient la cascade de signalisation. Des dysfonctions dans la signalisation médiée par les GPCRs sont corrélées avec de nombreuses maladies comme le diabète, des déficiences immunes et nerveuses, ainsi que le cancer. Puisque la voie de signalisation s'active et se désactive, les protéines G (Gs, Gq/11, G12/13 et Gi/o) ont été un sujet de recherche attrayant pendant des décennies. Une protéine G hétérotrimérique est composée de trois sous-unités, la sous-unité a associée au nucléotide guanine, ainsi que les sous-unités ß et y. En général, la durée du signal est déterminée par le temps de demi-vie des sous-unités Ga activées (Ga liées au GTP). Identifier de nouveaux partenaires de communication des sous-unités Ga se révèle être un moyen attractif de comprendre la machinerie de la signalisation par les GPCRs. Dans notre laboratoire nous nous sommes concentrés principalement sur Gao qui est exprimée de manière abondante dans le système nerveux. Nous présentons ici deux nouveaux partenaires qui interagissent avec Gao chez la drosophile: Dhit et Kermit, qui ont été identifiés respectivement par la méthode du yeast two-hybrid et par criblage génétique. Dhit est caractérisé par une petite taille, avec un domaine RGS conservé et un motif N- terminal riche en cystéines. Le domaine RGS contient une activité GAP (GTPase activating protein) pour les protéines G. Toutefois, nous avons découvert que Dhit exerce non seulement une activité GAP mais aussi une activité GDI (guanine nucleotide dissociation inhibitor) à l'égard de Gao. Cette activité GDI inattendue est préservée dans RGS19 - un homologue de Dhit chez les mammifères. Des expériences supplémentaires ont confirmé l'activité GDI de Dhit et de RGS19 chez Drosophila melanogaster et les modèles cellulaires mammifères. Par conséquent, nous proposons que Dhit et ses homologues mammifères modulent la signalisation GPCR par une double suppression des sous-unités Ga - suppression de leur nucléotide d'échange avec le GTP et une accélération dans leur hydrolyse du GTP. Kermit/GIPC a été premièrement identifié comme un partenaire de liaison de RGS19 dans le criblage par yeast two-hybrid. Au lieu d'interagir avec l'homologue chez la drosophile de RGS19 (Dhit), Kermit se lie à Gao in vivo et in vitro. La conséquence fonctionnelle de l'interaction Kermit/Gao est la régulation de la localisation de Vang, un des composants essentiel de la polarité planaire cellulaire, à la membrane apicale. Globalement, mon travail a démontré l'action de Gao avec ses deux partenaires d'interaction dans la voie de signalisation médiée par Gao. La compréhension de la signalisation par les GPCRs incluant Gao et ses régulateurs ou effecteurs aboutira à mettre en lumière de futurs axes dans la recherche pharmacologique.