Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Forming efficient networks

In this paper, we suggest a simple sequential mechanism whose subgame perfect equilibria give rise to efficient networks. Moreover, the payoffs received by the agents coincide with their Shapley value in an appropriately defined cooperative game.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Behavior study of pipes after forming, coating and bending

A study of the main types of coatings and its processes that modern industry commonly apply to prevent to the corrosion due to the environmental effects to energetic market pipelines have been done. Extracting main time and temperature range values, coating heat treatment recreation have been applied to x65 pipelines steel grade samples obtained from a pipe which was formed using UOE forming process. Experimental tensile tests and Charpy V‐Notch Impact test have been carried out for a deeply knowledge of the influence on the steel once this recreations are applied. The Yield Strength and toughness have been improved despite lower values in rupture strain and ductile‐brittle temperature transition have been obtained. Finite Element Method have been applied to simulate the entirely pipe cold bending process to predict the mechanical properties and behaviour of the pipe made from x65 steel grade under different conditions.

The Molecular Basis for Antimicrobial Activity of Pore-Forming Cyclic Peptides

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1–2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides

Disseny dels elements constitutius per adaptar un centre de mecanitzat de control numèric a la tecnologia Incremental Sheet Forming (ISF)

Un dels processos tradicionals de conformació de xapa més utilitzats a nivell industrial, degut a la seva rapidesa d’operació i la seva maduresa del procés, és l’embotició. Per a dur a terme aquest procés és necessària la construcció d’uns utillatges (matriu i punxó) per a cada tipus de producte. Aquests utillatges estan fabricats amb materials altament resistents ja que han de poder suportar càrregues molt importants durant la deformació. A més, s’han de garantir unes precisions dimensionals molt bones per tal d’evitar el xoc entre les dues parts de l’utillatge i per a assegurar que la peça obtinguda tingui les dimensions desitjades. Aquest fet implica que el procés d’obtenció de les matrius i punxons tingui un cost molt elevat i per a amortitzar-lo és necessari que la producció sigui en massa.L’objecte d’aquest projecte és adaptar el centre de mecanitzat Kondia HS1000 per tal de poder dur a terme recerca bàsica de la tecnologia de conformat incremental de xapa (ISF) en el Grup de Recerca en Enginyeria del Producte, Procés i Producció (GREP)

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.

External fluctuations in a pattern-forming instability

The effect of external fluctuations on the formation of spatial patterns is analyzed by means of a stochastic Swift-Hohenberg model with multiplicative space-correlated noise. Numerical simulations in two dimensions show a shift of the bifurcation point controlled by the intensity of the multiplicative noise. This shift takes place in the ordering direction (i.e., produces patterns), but its magnitude decreases with that of the noise correlation length. Analytical arguments are presented to explain these facts.

Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli

In this review we highlight recent work that has increased our understanding of the distribution of Shiga toxin-converting phages that can be detected as free phage particles, independently of Shiga toxin-producing bacteria (STEC). Stx phages are a quite diverse group of temperate phages that can be found in their prophage state inserted within the STEC chromosome, but can also be found as phages released from the cell after activation of their lytic cycle. They have been detected in extraintestinal environments such as water polluted with feces from humans or animals, food samples or even in stool samples of healthy individuals. The high persistence of phages to several inactivation conditions makes them suitable candidates for the successful mobilization of stx genes, possibly resulting in the genes reaching a new bacterial genomic background by means of transduction, where ultimately they may be expressed, leading to Stx production. Besides the obvious fact that Stx phages circulating between bacteria can be, and probably are, involved in the emergence of new STEC strains, we review here other possible ways in which free Stx phages could interfere with the detection of STEC in a given sample by current laboratory methods and how to avoid such interference.

Lipid rafts act as specialised domains for tetanus toxin binding and internalisation in neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.

An Analysis of Eye Scanpath Entropy in a Progressively Forming Virtual Environment

A sign of presence in virtual environments is that people respond to situations and events as if they were real, where response may be considered at many different levels, ranging from unconscious physiological responses through to overt behavior,emotions, and thoughts. In this paper we consider two responses that gave different indications of the onset of presence in a gradually forming environment. Two aspects of the response of people to an immersive virtual environment were recorded: their eye scanpath, and their skin conductance response (SCR). The scenario was formed over a period of 2 min, by introducing an increasing number of its polygons in random order in a head-tracked head-mounted display. For one group of experimental participants (n 8) the environment formed into one in which they found themselves standing on top of a 3 m high column. For a second group of participants (n 6) the environment was otherwise the same except that the column was only 1 cm high, so that they would be standing at normal ground level. For a third group of participants (n 14) the polygons never formed into a meaningful environment. The participants who stood on top of the tall column exhibited a significant decrease in entropy of the eye scanpath and an increase in the number of SCR by 99 s into the scenario, at a time when only 65% of the polygons had been displayed. The ground level participants exhibited a similar decrease in scanpath entropy, but not the increase in SCR. The random scenario grouping did not exhibit this decrease in eye scanpath entropy. A drop in scanpath entropy indicates that the environment had cohered into a meaningful perception. An increase in the rate of SCR indicates the perception of an aversive stimulus. These results suggest that on these two dimensions (scanpath entropy and rate of SCR) participants were responding realistically to the scenario shown in the virtual environment. In addition, the response occurred well before the entire scenario had been displayed, suggesting that once a set of minimal cues exists within a scenario,it is enough to form a meaningful perception. Moreover, at the level of the sympathetic nervous system, the participants who were standing on top of the column exhibited arousal as if their experience might be real. This is an important practical aspect of the concept of presence.

The star forming region Monoceros R2 as a gamma-ray source

After the release of the gamma-ray source catalog produced by the Fermi satellite during its first two years of operation, a significant fraction of sources still remain unassociated at lower energies. In addition to well-known high-energy emitters (pulsars, blazars, supernova remnants, etc.), theoretical expectations predict new classes of gamma-ray sources. In particular, gamma-ray emission could be associated with some of the early phases of stellar evolution, but this interesting possibility is still poorly understood. Aims: The aim of this paper is to assess the possibility of the Fermi gamma-ray source 2FGL J0607.5-0618c being associated with the massive star forming region Monoceros R2. Methods: A multi-wavelength analysis of the Monoceros R2 region is carried out using archival data at radio, infrared, X-ray, and gamma-ray wavelengths. The resulting observational properties are used to estimate the physical parameters needed to test the different physical scenarios. Results: We confirm the 2FGL J0607.5-0618c detection with improved confidence over the Fermi two-year catalog. We find that a combined effect of the multiple young stellar objects in Monoceros R2 is a viable picture for the nature of the source.

Combining genetic and demographic data for the conservation of a Mediterranean habitat-forming species

The integration of ecological and evolutionary data is highly valuable for conservation planning. However, it has been rarely used in the marine realm, where the adequate design of marine protected areas (MPAs) is urgently needed. Here, we examined the interacting processes underlying the patterns of genetic structure and demographic strucuture of a highly vulnerable Mediterranean habitat-forming species (i.e. Paramuricea clavata (Risso, 1826)), with particular emphasis on the processes of contemporary dispersal, genetic drift, and colonization of a new population. Isolation by distance and genetic discontinuities were found, and three genetic clusters were detected; each submitted to variations in the relative impact of drift and gene flow. No founder effect was found in the new population. The interplay of ecology and evolution revealed that drift is strongly impacting the smallest, most isolated populations, where partial mortality of individuals was highest. Moreover, the eco-evolutionary analyses entailed important conservation implications for P. clavata. Our study supports the inclusion of habitat-forming organisms in the design of MPAs and highlights the need to account for genetic drift in the development of MPAs. Moreover, it reinforces the importance of integrating genetic and demographic data in marine conservation.