Optimization and assessment of a novel gastric electrode anchoring system designed to be implanted by minimally invasive surgery.

A novel electrode anchoring design and its implantation procedure, aiming for a minimally invasive solution for gastric electrical stimulation, are presented. The system comprises an anchor made of a flexible body embedding two needle-shaped electrodes. The electrodes can easily switch from a parallel position - to pierce the stomach - to a diverging position - enabling them to remain firmly anchored into the muscular layer of the stomach. Key device parameters governing anchoring stability were assessed on a traction test bench, and optimal values were derived. The device was then implanted in six dogs by open surgery to assess its anchoring durability in vivo. Computed tomography images showed that the electrodes remained well placed within the dogs' gastric wall over the entire assessment period (more than one year). Finally, a prototype of a surgical tool for the minimally invasive device placement was manufactured, and the anchoring procedure was tested on a dog cadaver, providing the proof of concept of the minimally invasive implantation procedure. The use of our electrode anchoring system in long-term gastric electrical stimulation is promising in terms of implantation stability (the anchor withstands a force up to 0.81 N), durability (the anchor remains onto the stomach over one year) and minimal invasiveness of the procedure (the diameter of the percutaneous access is smaller than 12 mm). Moreover, the proposed design could have clinical applications in other hollow organs, such as the urinary bladder.

Use of bifunctional hybrid beta-lactamases for epitope mapping and immunoassay development.

Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis beta-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random gene fragments. The procedure presented in this paper allows the selection of large infectious phage libraries with high diversity and efficient beta-lactamase activities. A useful aspect of the proposed technique results from the possibility of using the beta-lactamase activity carried by phages to evaluate the proportion of immobilised phages during the successive enrichment steps of the library or competition experiments with the selected phages. Another advantage of the technique derives from the fact that the epitope is selected as a bifunctional hybrid protein, which can be overproduced and purified. The resulting recombinant protein associates an epitope with a specific and efficient enzymatic activity. This constitutes an original tool for immunoassay development. A virus influenza hemagglutinin (HA1)-gene fragment library has been generated with this system and used to identify a linear epitope.

Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP.

The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco). The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).

Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies.

Haydon and Guest (Haydon, D. J, and Guest, J. R. (1991) FEMS Microbiol. Lett. 63, 291-295) first described the helix-turn-helix GntR family of bacterial regulators. They presented them as transcription factors sharing a similar N-terminal DNA-binding (d-b) domain, but they observed near-maximal divergence in the C-terminal effector-binding and oligomerization (E-b/O) domain. To elucidate this C-terminal heterogeneity, structural, phylogenetic, and functional analyses were performed on a family that now comprises about 270 members. Our comparative study first focused on the C-terminal E-b/O domains and next on DNA-binding domains and palindromic operator sequences, has classified the GntR members into four subfamilies that we called FadR, HutC, MocR, and YtrA. Among these subfamilies a degree of similarity of about 55% was observed throughout the entire sequence. Structure/function associations were highlighted although they were not absolutely stringent. The consensus sequences deduced for the DNA-binding domain were slightly different for each subfamily, suggesting that fusion between the D-b and E-b/O domains have occurred separately, with each subfamily having its own D-b domain ancestor. Moreover, the compilation of the known or predicted palindromic cis-acting elements has highlighted different operator sequences according to our subfamily subdivision. The observed C-terminal E-b/O domain heterogeneity was therefore reflected on the DNA-binding domain and on the cis-acting elements, suggesting the existence of a tight link between the three regions involved in the regulating process.