Comparative transcriptomics of early dipteran development

BACKGROUND: Modern sequencing technologies have massively increased the amount of data available for comparative genomics. Whole-transcriptome shotgun sequencing (RNA-seq) provides a powerful basis for comparative studies. In particular, this approach holds great promise for emerging model species in fields such as evolutionary developmental biology (evo-devo). RESULTS: We have sequenced early embryonic transcriptomes of two non-drosophilid dipteran species: the moth midge Clogmia albipunctata, and the scuttle fly Megaselia abdita. Our analysis includes a third, published, transcriptome for the hoverfly Episyrphus balteatus. These emerging models for comparative developmental studies close an important phylogenetic gap between Drosophila melanogaster and other insect model systems. In this paper, we provide a comparative analysis of early embryonic transcriptomes across species, and use our data for a phylogenomic re-evaluation of dipteran phylogenetic relationships. CONCLUSIONS: We show how comparative transcriptomics can be used to create useful resources for evo-devo, and to investigate phylogenetic relationships. Our results demonstrate that de novo assembly of short (Illumina) reads yields high-quality, high-coverage transcriptomic data sets. We use these data to investigate deep dipteran phylogenetic relationships. Our results, based on a concatenation of 160 orthologous genes, provide support for the traditional view of Clogmia being the sister group of Brachycera (Megaselia, Episyrphus, Drosophila), rather than that of Culicomorpha (which includes mosquitoes and blackflies).

Genomics of wine yeast: functional analysis of new and variable genes

Projecte de recerca elaborat a partir d’una estada a l’Institut National de la Recherche Agronomique, França, entre 2007 i 2009. Saccharomyces cerevisiae ha estat el llevat utilitzat durant mil.lenis en l'elaboració de vins. Tot i així, es té poc coneixement sobre les pressions de selecció que han actuat en la modelització del genoma dels llevats vínics. S’ha seqüenciat el genoma d'una soca vínica comercial, EC1118, obtenint 31 supercontigs que cobreixen el 97% del genoma de la soca de referència, S288c. S’ha trobat que el genoma de la soca vínica es diferencia bàsicament en la possessió de 3 regions úniques que contenen 34 gens implicats en funcions claus per al procés fermentatiu. A banda, s’han dut a terme estudis de filogènia i synteny (ordre dels gens) que mostren que una d'aquestes tres regions és pròxima a una espècie relacionada amb el gènere Saccharomyces, mentre que les altres dos regions tenen un origen no-Saccharomyces. S’ha identificat mitjançant PCR i seqüenciació a Zygosaccharomyces bailii, una espècie contaminant de les fermentacions víniques, com a espècie donadora d'una de les dues regions. Les hibridacions naturals entre soques de diferents espècies dins del grup Saccharomyces sensu stricto ja han estat descrites. El treball és el primer que presenta hibridacions entre espècies Saccharomyces i no-Saccharomyces (Z. bailii, en aquest cas). També s’assenyala que les noves regions es troben freqüent i diferencialment presents entre els clades de S. cerevisiae, trobant-se de manera gairebé exclusiva en el grup de les soques víniques, suggerint que es tracta d'una adquisició recent de transferència gènica. En general, les dades demostren que el genoma de les soques víniques pateix una constant remodelació mitjançant l'adquisició de gens exògens. Els resultats suggereixen que aquests processos estan afavorits per la proximitat ecològica i estan implicats en l'adaptació molecular de les soques víniques a les condicions d'elevada concentració en sucres, poc nitrogen i elevades concentracions en etanol.

Factors de fracàs de la ventilació mecànica no invasiva en malalts crítics amb insuficiència respiratòria aguda hipoxèmica de novo

Resum: Introducció: En la hipoxemia de novo la indicació de la VNI és controvertida. Sembla que el fracàs de la VNI s’associa a major mortalitat en aquests malalts. Objectiu: Identificar possibles factors predictors de fracàs de la VNI. Pacients i mètodes: Estudi observacional retrospectiu de 35 malalts ingressats per hipoxemia de novo i que van rebre tractament de suport amb ventilació no invasiva. Resultats: En aquests malalts la VNi va fracasar més del 50%, sobretot en malalts amb SDRA i en els més hipoxèmics. Els malalts que van fracassar van tenir major mortalitat, més infeccions nosocomials respiratòries i més dies d’ingrés a UCI.

The Voting Practice of the Fifteen in the UN General Assembly: Convergence and Divergence

The EU has, since the early days of the Community, had the ambition to speak with ‘a single voice’ in international fora, in particular in the United Nations’ General Assembly. This aspiration, which has become more pronounced since the inauguration of the CFSP, has not always been easy to achieve due to domestic or international level factors affecting the EU member states. However, in the last decade there has been a dramatic increase in convergence in the Fifteen’s voting record. This paper contemplates the underlying reasons for such a convergence

Assembly and disassembly of the Golgi Complex: two prodesses arranged in a cis to trans direction

We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt-1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans-Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans-Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae.

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4¿dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Viral self-assembly as a thermodynamic process

The protein shells, or capsids, of nearly all spherelike viruses adopt icosahedral symmetry. In the present Letter, we propose a statistical thermodynamic model for viral self-assembly. We find that icosahedral symmetry is not expected for viral capsids constructed from structurally identical protein subunits and that this symmetry requires (at least) two internal switching configurations of the protein. Our results indicate that icosahedral symmetry is not a generic consequence of free energy minimization but requires optimization of internal structural parameters of the capsid proteins

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4¿dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.

Colloidal assembly on magnetically vibrated stripes

We investigate the collective organization of paramagnetic colloidal particles externally driven above the periodic stripes of a uniaxial ferrimagnetic garnet film. An external field modulation induces vibration of the stripe walls and produces random motion of the particles. Defects in the stripe pattern break the symmetry of the potential and favor particle nucleation into large clusters above a critical density. Mismatch between particle size and pattern wavelength generates assemblies with different morphological order. At even higher field strengths, repulsive dipolar interactions between the particles induce cluster melting. We propose a novel approach to generate and externally control a variety of colloidal assemblies.

Self-organizing propagation patterns from dynamic self-assembly in monolayers

Propagation of localized orientational waves, as imaged by Brewster angle microscopy, is induced by low intensity linearly polarized light inside axisymmetric smectic-C confined domains in a photosensitive molecular thin film at the air/water interface (Langmuir monolayer). Results from numerical simulations of a model that couples photoreorientational effects and long-range elastic forces are presented. Differences are stressed between our scenario and the paradigmatic wave phenomena in excitable chemical media.

Self-organizing propagation patterns from dynamic self-assembly in monolayers

Propagation of localized orientational waves, as imaged by Brewster angle microscopy, is induced by low intensity linearly polarized light inside axisymmetric smectic-C confined domains in a photosensitive molecular thin film at the air/water interface (Langmuir monolayer). Results from numerical simulations of a model that couples photoreorientational effects and long-range elastic forces are presented. Differences are stressed between our scenario and the paradigmatic wave phenomena in excitable chemical media.

Model studies on the synthesis of madangamine alkaloids. Assembly of the macrocyclic rings

Using simplified model derivatives, the assembly of the macrocyclic rings of madangamines, including the 13- and 14-membered D rings of madangamines C-E, the all-cis-triunsaturated 15-membered D ring of madangamine A, and the (Z,Z)-unsaturated 11-membered E ring common to madangamines A-E, has been studied.