Coupling of a targeting peptide to plasmid DNA using a new type of padlock oligonucleotide

We have recently described a new method for attaching padlock oligonucleotides to supercoiled plasmid DNA at specific sequences. A variant of this method has been developed in order to allow the coupling of targeting moieties to plasmids using a convenient strategy. After sequence-specific winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, the extremities of a triplex-forming oligonucleotide hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a hairpin oligonucleotide using T4 DNA ligase. Any targeting moiety may be attached to the hairpin oligonucleotide. This strategy was used to attach an NLS peptide to a luciferase-expressing plasmid. Despite the presence of the padlock oligonucleotide, the reporter gene was efficiently expressed after transfection of the plasmid in HeLa or T24 cells, using either cationic lipids or cationic polymers as transfecting agents. However, no increase in gene expression could be observed as a result of peptide attachment. Nevertheless, the coupling strategy described in this paper may find applications as a tool for plasmid functionalization in other targeting experiments, and may lead to the development of improved vectors for gene therapy.     

Structure and function of the C-terminal domain of methionyl-tRNA synthetase

The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.     

Vibration analysis on incubating eggs and its relation to embryonic development

Coucke (1998) was the first to use acoustic resonance analysis to monitor embryo development in chicken eggs. He remarked that at around 100 hours of incubation, the course of the resonant frequency and damping changed abruptly in the case of fertile eggs. He also showed that these changes were related to a physiologic event during early embryonic development. The objective of our study is to monitor the course of the vibration parameters during the early incubation of chicken eggs and to relate these changes to egg and embryo characteristics. A total of 72 Hybro eggs were incubated vertically in a small incubator at standard conditions. Several egg parameters were measured before incubation. During the early stages of incubation the vibration behavior of these eggs was monitored. The time at which the damping of the vibration suddenly changed, the diameter of the eggs and their interaction were found to be significant explanatory variables in order to predict hatching time. A correlation coefficient r of 0.72 was obtained.     

A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris

Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.     

Assessment of embryonic growth in chicken eggs by means of visible transmission spectroscopy

During this work, it was investigated whether spectral measurements can be used to monitor embryonic growth. An experiment was conducted in which both the transmission spectra and embryonic weight were determined on 240 eggs (Cobb, 37 weeks) between Day 5 and Day 10 of incubation. The spectral data were linked to embryonic weight by means of a partial least squares analysis. Different preprocessing procedures were compared during this work, that is, smoothing, multiplicative scatter correction (MSC), and first‐ and second‐order derivative. Compared to the remainder of the preprocessing procedures, MSC leads to a considerable improvement of the prediction capability of the embryonic weight. The ratio of performance to deviation obtained for the MSC spectra equaled 4.5 indicating that a very accurate prediction of embryonic weight is feasible based on the VIS/NIR transmission measurements. Important regions for the prediction are situated around 685–740 nm. It is suggested that the spectral changes in these spectral regions result from the displacement of carotenoids from the yolk into the blood circuitry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation

BackgroundSeveral adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation.ResultsIn the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells.ConclusionsOverall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular/cellular effects associated with muscle repair and the clinical efficacy of MuStem cell-based therapy.     

Topology Analysis of Social Networks Extracted from Literature

In a world where complex networks are an increasingly important part of science, it is interesting to question how the new reading of social realities they provide applies to our cultural background and in particular, popular culture. Are authors of successful novels able to reproduce social networks faithful to the ones found in reality? Is there any common trend connecting an author’s oeuvre, or a genre of fiction? Such an analysis could provide new insight on how we, as a culture, perceive human interactions and consume media. The purpose of the work presented in this paper is to define the signature of a novel’s story based on the topological analysis of its social network of characters. For this purpose, an automated tool was built that analyses the dialogs in novels, identifies characters and computes their relationships in a time-dependent manner in order to assess the network’s evolution over the course of the story.     

Infectious Chikungunya Virus in the Saliva of Mice,Monkeys and Humans

Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7^-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7^-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7^-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7^-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.