Real-time in vivo ROS monitoring with luminescent nanoparticles reveals skin inflammation dynamics.

Reactive oxygen species (ROS) are key regulators in numerous pathological contexts, including cancer or inflammation. Their role is complex, which justifies the need for methods enabling their quantitative and time-resolved monitoring in vivo, in the perspective to profile tissues of individual patients. However, current ROS detection methods do not provide these features. Here, we propose a new method based on the imaging of lanthanide-ion nanoparticles (GdVO4:Eu), whose photoluminescence is modulated by the surrounding ROS concentration. We monitored their luminescence after intradermic injection in a mouse ear submitted to an inflammation-inducing topical stimulus. Based on this approach, we quantified the ROS concentration after inflammation induction and identified a two-step kinetics of ROS production, which may be attributed to the response of resident immune cells and their further recruitment at the inflammation locus.

Exploitation of the chick embryo chorioallantoic membrane (CAM) as a platform for anti-metastatic drug testing.

The establishment of clinically relevant models for tumor metastasis and drug testing is a major challenge in cancer research. Here we report a physiologically relevant assay enabling quantitative analysis of metastatic capacity of tumor cells following implantation into the chorioallantoic membrane (CAM). Engraftment of as few as 103 non-small cell lung cancer (NSCLC) and prostate cancer (PCa) cell lines was sufficient for both primary tumor and metastasis formation. Standard 2D-imaging as well as 3D optical tomography imaging were used for the detection of fluorescent metastatic foci in the chick embryo. H2228- and H1975-initiated metastases were confirmed by genomic analysis. We quantified the inhibitory effect of docetaxel on LNCaP, and that of cisplatin on A549- and H1299-initiated metastatic growths. The CAM assay also mimicked the sensitivity of ALK-rearranged H2228 and EGFR-mutated H1975 NSCLC cells to tyrosine kinase inhibitors crizotinib and gefitinib respectively, as well as sensitivity of LNCaP cells to androgen-dependent enzalutamide therapy. The assay was suggested to reconstitute the bone metastatic tropism of PCa cells. We show that the CAM chick embryo model may be a powerful preclinical platform for testing and targeting of the metastatic capacity of cancer cells.

Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy.

BACKGROUND: Dmdmdx , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. METHODS: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. RESULTS: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. CONCLUSION: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo.

In the central nervous system (CNS), there are innate obstacles to the modification of neurons: their relative low abundance versus glia and oligodendrocytes, the inaccessibility of certain target populations, and the volume one can inject safely. Our aim in this study was to characterize the in vivo efficacy of a novel viral vector derived from a canine adenovirus (CAV-2). Here we show that CAV-2 preferentially transduced i) rat olfactory sensory neurons; ii) rodent CNS neurons in vitro and in vivo; and, more clinically relevant, iii) neurons in organotypic slices of human cortical brain. CAV-2 also showed a high disposition for retrograde axonal transport in vivo. We examined the molecular basis of neuronal targeting by CAV-2 and suggest that due to CAR (coxsackie adenovirus receptor) expression on neuronal cells-and not oligodendrocytes, glia, myofibers, and nasal epithelial cells-CAV-2 vectors transduced neurons preferentially in these diverse tissues.

Application of flow cytometry to rapid microbial analysis in food and drinks industries.

In food and drinks industries, the time required for conventional tests can lead to substantial delays in product release to the market. Flow cytometry (FCM) has been used in conjunction with viability markers for rapid counting of yeast, mould and bacterial cells in food products. A single-parameter flow cytometer has proved applicable to the rapid detection of low numbers of microbial contaminants in finished products. The excellent correlation between FCM results and product quality shelf-life expiry date has allowed the establishment of realistic quality control criteria for rapid positive release of product. Used for the monitoring of microbial biomass during manufacturing processes, flow cytometry allowed a direct assessment of bacterial growth. The reproducibility of the results and the proven correlation with standard plate count method obtained in industrial conditions make FCM a good predictive method for product and process quality control.