In vivo gene transfer into the ocular ciliary muscle mediated by ultrasound and microbubbles.

This study aimed to assess application of ultrasound (US) combined with microbubbles (MB) to transfect the ciliary muscle of rat eyes. Reporter DNA plasmids encoding for Gaussia luciferase, β-galactosidase or the green fluorescent protein (GFP), alone or mixed with 50% Artison MB, were injected into the ciliary muscle, with or without US exposure (US set at 1 MHz, 2 W/cm(2), 50% duty cycle for 2 min). Luciferase activity was measured in ocular fluids at 7 and 30 days after sonoporation. At 1 week, the US+MB treatment showed a significant increase in luminescence compared with control eyes, injected with plasmid only, with or without MB (×2.6), and, reporter proteins were localized in the ciliary muscle by histochemical analysis. At 1 month, a significant decrease in luciferase activity was observed in all groups. A rise in lens and ciliary muscle temperature was measured during the procedure but did not result in any observable or microscopic damages at 1 and 8 days. The feasibility to transfer gene into the ciliary muscle by US and MB suggests that sonoporation may allow intraocular production of proteins for the treatment of inflammatory, angiogenic and/or degenerative retinal diseases.

A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin.

BACKGROUND: The activity of the renin-angiotensin system is usually evaluated as plasma renin activity (PRA, ngAI/ml per h) but the reproducibility of this enzymatic assay is notoriously scarce. We compared the inter and intralaboratory reproducibilities of PRA with those of a new automated chemiluminescent assay, which allows the direct quantification of immunoreactive renin [chemiluminescent immunoreactive renin (CLIR), microU/ml]. METHODS: Aliquots from six pool plasmas of patients with very low to very high PRA levels were measured in 12 centres with both the enzymatic and the direct assays. The same methods were applied to three control plasma preparations with known renin content. RESULTS: In pool plasmas, mean PRA values ranged from 0.14 +/- 0.08 to 18.9 +/- 4.1 ngAI/ml per h, whereas those of CLIR ranged from 4.2 +/- 1.7 to 436 +/- 47 microU/ml. In control plasmas, mean values of PRA and of CLIR were always within the expected range. Overall, there was a significant correlation between the two methods (r = 0.73, P < 0.01). Similar correlations were found in plasmas subdivided in those with low, intermediate and high PRA. However, the coefficients of variation among laboratories found for PRA were always higher than those of CLIR, ranging from 59.4 to 17.1% for PRA, and from 41.0 to 10.7% for CLIR (P < 0.01). Also, the mean intralaboratory variability was higher for PRA than for CLIR, being respectively, 8.5 and 4.5% (P < 0.01). CONCLUSION: The measurement of renin with the chemiluminescent method is a reliable alternative to PRA, having the advantage of a superior inter and intralaboratory reproducibility.

Nanoparticles for fingermark detection: an insight into the reaction mechanism

This publication presents one of the first uses of silicon oxide nanoparticles to detect fingermarks. The study is not confined to showing successful detection of fingermarks, but is focused on understanding the mechanisms involved in the fingermark detection process. To gain such an understanding, various chemical groups are grafted onto the nanoparticle surface, and parameters such as the pH of the solutions or zeta potential are varied to study their influence on the detection. An electrostatic interaction has been the generally accepted hypothesis of interaction between nanoparticles and fingermarks, but the results of this research challenge that hypothesis, showing that the interaction is chemically driven. Carboxyl groups grafted onto the nanoparticle surfaces react with amine groups of the fingermark secretion. This formation of amide linkage between carboxyl and amine groups has further been favoured by catalyzing the reaction with a compound of diimide type. The research strategy adopted here ought to be applicable to all detection techniques using nanoparticles. For most of them the nature of the interaction remains poorly understood.

Laser-Ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8x106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 mu l) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 mu s instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemining from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.

Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.

For the past 10 years, mini-host models and in particular the greater wax moth Galleria mellonella have tended to become a surrogate for murine models of fungal infection mainly due to cost, ethical constraints and ease of use. Thus, methods to better assess the fungal pathogenesis in G. mellonella need to be developed. In this study, we implemented the detection of Candida albicans cells expressing the Gaussia princeps luciferase in its cell wall in infected larvae of G. mellonella. We demonstrated that detection and quantification of luminescence in the pulp of infected larvae is a reliable method to perform drug efficacy and C. albicans virulence assays as compared to fungal burden assay. Since the linearity of the bioluminescent signal, as compared to the CFU counts, has a correlation of R(2) = 0.62 and that this method is twice faster and less labor intensive than classical fungal burden assays, it could be applied to large scale studies. We next visualized and followed C. albicans infection in living G. mellonella larvae using a non-toxic and water-soluble coelenterazine formulation and a CCD camera that is commonly used for chemoluminescence signal detection. This work allowed us to follow for the first time C. albicans course of infection in G. mellonella during 4 days.

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.