Continuous microcarrier-based cell culture in a benchtop microfluidic bioreactor.

Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.

Improvement of yeast biochip sensitivity using multilayer inorganic sol-gel substrates.

Today, microarray fluorescence detection is still limited because a great proportion of hybrids remain undetectable. In this paper we describe sol-gel optical multilayers (stacks of low- and high-index layers) deposited on glass slides which increase the fluorescence of DNA microarrays and favour the detection of fluorescent targets. An alternative to the expensive and time-consuming physical vapour deposition technology is proposed. It is a low-cost sol-gel coating of glass slides, each layer being made by "dipping" (alternatively in SiO2 or TiO2 solutions), "draining and drying". After the selection of the best surface layer of the substrates, the multilayer mirrors modelled for one (Cy3) or two (Cy3 and Cy5) fluorophores are spotted with a series of Yeast probes and compared to similar microarrays on standard glass slides through hybridisation experiments. The fluorescence images of the mirrors show increased signals for all the probes. The enhancement factors determined for Cy3 and for Cy3/Cy5 mirrors (10-12 and 4-5, respectively) are consistent with the initial modelling. This allows the assessment of the basal expression levels of Yeast low-expressed genes. Moreover, these substrates show a noticeable increase in sensitivity for induction/repression ratio measurements in differential gene expression experiments. So, they could be considered as promising tools for the analysis of small biological samples.

Multiple wavelength fluorescence enhancement on glass substrates for biochip and cell analyses.

In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve the sensitivity of the fluorescence detection of both common fluorophores Cy3 and Cy5 in DNA microarrays and fluorescent cell analyses, we have designed a multi layer mirror with alternative thin layers of SiO2 and HfO2. This mirror (MOTL) prevents fluorescence absorption, allows the simultaneous enhancement of the fluorescence signals and increases the dynamic range of the slides. Using MOTL slides, Cy3 and Cy5 intensities are enhanced by 5-8-fold, consequently, the fluorescence analysis becomes easier and should allow the detection of low copy number genes or weakly fluorescent cells. With the same approach, other multiple optical thin layer slides could be designed for other series of fluorophores, extending the field of their applications.

Both TCR alpha and TCR delta chain diversity are regulated during thymic ontogeny.

TCRalpha and TCRdelta chains are coded by a common genetic locus using a single set of V gene segments (ADV segments). This article addresses the question of regulation of the use of the ADV segments by the TCRalpha and TCRdelta chains. Using both qualitative and quantitative analyses we have studied the use of 23 ADV gene families as part of TCRalpha and TCRdelta transcripts. A number of previously undetected rearrangement and transcription events are described, indicating that the intrathymic TCRdelta repertoire is much more diverse than previously supposed. Repertoire analysis at several developmental time points allowed the description of regulated waves of ADV gene use, not only for TCRdelta chains, but also for TCRalpha chains, during thymic ontogeny. Control of these waves appears to be linked directly to the ADV segments and their local chromatin environment, which may change over the course of T cell differentiation.

Expression hybridization assays combining cDNAs from firefly and Renilla luciferases as labels for simultaneous determination of two target sequences.

Two cDNAs encoding firefly luciferase (FLuc) and Renilla luciferase (RLuc) were used as labels for the development of a microtiter well-based expression hybridization assay that allows simultaneous determination of two target DNA sequences. The target DNAs were denatured and hybridized with specific capture and detection probes. One detection probe was biotinylated while the other was tailed with poly(dT). The hybrids were reacted with a streptavidin-FLuc DNA complex and a poly(dA)-tailed RLuc DNA, respectively. Subsequently, the cDNA labels were expressed in vitro simultaneously and independently in the same transcription/translation reaction mixture. The activities of generated firefly and Renilla luciferases were co-determined in the same sample based on the differential requirements of their characteristic bioluminescent reactions for magnesium ions.

Effects of smoking on periodontal health: a review.

Cigarette smoking is a risk factor for several diseases, and recent evidence strongly suggests an adverse effect on periodontal health. Nevertheless, the nature of the relationship between smoking and periodontal disease is not clear. Smoking causes defects in neutrophil function, impairs inflammatory and immune responses to periodontal pathogens, and exerts both systemic and local effects. Smoking is associated with an increased rate of periodontal disease in terms of alveolar bone loss and attachment loss, as well as pocket formation. Nicotine, the major component of cigarette smoke, may weaken host defenses to the bacterial invasion induced by plaque.

[Smoking and periodontal health]. / Le tabagisme et la santé parodontale.

The nature of the relationship between smoking and periodontal disease is not clear. However, from the information available today, the evidence, when carefully weighted, strongly suggests that smoking exerts a substantial and detrimental effect on periodontal health and disease. It is associated with an increased disease rate in terms of periodontal bone loss, attachment loss as well as periodontal pocket formation and it seems to worsen the host's defense by means of its major metabolite, nicotine. Nevertheless, new, sensitive and adequate investigations should be developed and performed in order to better explain the pathogenic mechanisms.