Impact of Anesthetics on Immune Functions in a Rat Model of Vagus Nerve Stimulation

Vagus nerve stimulation (VNS) has been successfully performed in animals for the treatment of different experimental models of inflammation. The anti-inflammatory effect of VNS involves the release of acetylcholine by vagus nerve efferent fibers inhibiting pro-inflammatory cytokines (e.g. TNF-α) produced by macrophages. Moreover, it has recently been demonstrated that splenic lymphocytic populations may also be involved. As anesthetics can modulate the inflammatory response, the current study evaluated the effect of two different anesthetics, isoflurane and pentobarbital, on splenic cellular and molecular parameters in a VNS rat model. Spleens were collected for the characterization of lymphocytes sub-populations by flow cytometry and quantification of cytokines secretion after in vitro activation. Different results were observed depending on the anesthetic used. The use of isoflurane displayed a non-specific effect of VNS characterized by a decrease of most splenic lymphocytes sub-populations studied, and also led to a significantly lower TNF-α secretion by splenocytes. However, the use of pentobarbital brought to light immune modifications in non-stimulated animals that were not observed with isoflurane, and also revealed a specific effect of VNS, notably at the level of T lymphocytes’ activation. These differences between the two anesthetics could be related to the anti-inflammatory properties of isoflurane. In conclusion, pentobarbital is more adapted than isoflurane in the study of the anti-inflammatory effect of VNS on an anesthetized rat model in that it allows more accurate monitoring of subtle immunomodulatory processes.     

Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems?

When, where and how is the head-tail axis of the embryo set up during development? These are such fundamental and intensely studied questions that one might expect them to have been answered long ago. Not so; we still understand very little about the cellular or molecular mechanisms that lead to the orderly arrangement of body elements along the head-tail axis in vertebrates. In this paper, we outline some of the major outstanding problems and controversies and try to identify some reasons why it has been so difficult to resolve this important issue.     

Impaired Leaf Litter Processing in Acidified Streams

Anthropogenic acidification in headwater streams is known to affect microbial assemblages involved in leaf litter breakdown. Far less is known about its potential effects on microbial enzyme activities. To assess the effects of acidification on microbial activities associated with decaying leaves, a 70-day litter bag experiment was conducted in headwater streams at six sites across an acidification gradient. The results revealed that microbial leaf decomposition was strongly and negatively correlated with total Al concentrations (r?=??0.99, p?<?0.001) and positively correlated with Ca²⁺ concentrations (r?=?0.94, p?=?0.005) and pH (r?=?0.93, p?=?0.008). Denaturing gradient gel electrophoresis analyses showed that microbial assemblages differed between non-impacted and impacted sites, whereas fungal biomass associated with decaying leaves was unaffected. The nutrient content of leaf detritus and ecoenzymatic activities of carbon (C), nitrogen (N) and phosphorus (P) acquisition revealed that N acquisition was unaltered, while P acquisition was significantly reduced across the acidification gradient. The P content of leaf litter was negatively correlated with total Al concentrations (r?=??0.94, p?<?0.01) and positively correlated with decomposition rates (r?=?0.95, p?<?0.01). This potential P limitation of microbial decomposers in impacted sites was confirmed by the particularly high turnover activity for phosphatase and imbalanced ratios between the ecoenzymatic activities of C and P acquisition. The toxic form of Al has well-known direct effects on aquatic biota under acidic conditions, but in this study, Al was found to also potentially affect microbially mediated leaf processing by interfering with the P cycle. These effects may in turn have repercussions on higher trophic levels and whole ecosystem functioning.     

Homology modeling and molecular dynamics simulations of the N-terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW-GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold-recognition techniques, we identified a fold compatible with the N-terminal domain of HMW-GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N-terminal domain of HMW-GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N-terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N-terminal domains of the y-type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22-44 and cysteines 10-55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N-terminal domain of Dy10, Nt-Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N-terminal domain has retained similar inhibitory functions.     

Insertion mutagenesis of Escherichiacoli GroEL

To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon ISlacZ/in. Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367. Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively. Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding. Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 degrees C whether or not GroES was co-expressed. A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90. By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible. Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.     

Biosensor for direct cell detection, quantification and analysis

Microarrays are promising tools for cell isolation and detection. However, they have yet to be widely applied in biology. This stems from a lack of demonstration of their sensitivity and compatibility with complex biological samples, and a lack of proof that their use does not induce aberrant cellular effects. Herein, we characterized and optimized a recently developed technology associating antibody microarrays with surface plasmon resonance imaging (SPRi). Using a murine macrophage cell line we demonstrate the binding specificity of our antibody-microarrays and the correlation between SPRi signals and both the number of bound cells, and the level of expression of cell surface markers. Confocal microscopy reveals that cell binding to the chip through antibody-antigen interactions underwent morphological changes reflecting the density of the relevant cell surface marker without affecting cell viability as shown by fluorescent microscopy. The detection threshold of the microarray-SPRi system is lowered 10-fold by applying a polyethylene oxide film to the gold surface of the chip. This increased sensitivity allows the detection of cells representing as little as 0.5% of a mixed population. The potential of this method is illustrated by two applications: characterization of ligand-cell receptor interactions, allowing determination of receptor specificity, and analysis of peripheral blood mononuclear cells, demonstrating the suitability of this tool for the analysis of complex biological samples.     

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants

Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.