How to use stem cells for repair in stroke patients.

Regenerative cell therapy is a promising therapeutic strategy in neurology, most notably to improve stroke recovery. Although tolerability and feasibility have apparently been validated, many questions remain as to what is the best type of cells to use, the best route and the post-stroke delay for administration. Two main strategies have currently emerged: intravenous injection of mesenchymal stem cells with systemic trophic support; and intracerebral grafting of neural stem cells with brain repair effects at the lesion site. Multicenter clinical trials have just begun and are starting to assess the efficacy of these treatments on functional recovery. However, experimental studies also need to be conducted in parallel to precisely identify the mechanisms of action regarding the pathophysiology of brain plasticity, notably when stroke occurs with comorbidities. Such studies should also evaluate the potential of cell grafting combined with injectable biomaterials.

Construction of a cytosolic firefly luciferase reporter cassette for use in PCR-mediated gene deletion and fusion in Saccharomyces cerevisiae.

Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(-SKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern.

Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea.

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca(2+) ions ([Ca(2+)](i)) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca(2+)](i). The ACh concentration-response curve indicated an apparent dissociation constant (K(d)) of 8 microM and a Hill coefficient of 1.0. Removing extracellular free Ca(2+) did not suppress the ACh-induced Ca(2+) responses suggesting an intracellular Ca(2+)-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca(2+) response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca(2+) responses similar to those observed with ACh, with an apparent K(d) of 10 microM and a Hill coefficient of 1.0. The cholinergic-induced Ca(2+)pirenzepine. Nicotine (10 to 100 microM) did not evoke Ca(2+) responses and the nicotinic antagonist curare (10 microM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.

Drug development in oncology assisted by noninvasive optical imaging.

Early and accurate detection of tumors, like the development of targeted treatments, is a major field of research in oncology. The generation of specific vectors, capable of transporting a drug or a contrast agent to the primary tumor site as well as to the remote (micro-) metastasis would be an asset for early diagnosis and cancer therapy. Our goal was to develop new treatments based on the use of tumor-targeted delivery of large biomolecules (DNA, siRNA, peptides, or nanoparticles), able to induce apoptosis while dodging the specific mechanisms developed by tumor cells to resist this programmed cell death. Nonetheless, the insufficient effectiveness of the vectorization systems is still a crucial issue. In this context, we generated new targeting vectors for drug and biomolecules delivery and developed several optical imaging systems for the follow-up and evaluation of these vectorization systems in live mice. Based on our recent work, we present a brief overview of how noninvasive optical imaging in small animals can accelerate the development of targeted therapeutics in oncology.