Blue light disrupts the circadian rhythm and create damage in skin cells.

On a daily basis, the skin is exposed to many environmental stressors and insults. Over a 24-h natural cycle, during the day, the skin is focused on protection; while at night, the skin is focused on repairing damage that occurred during daytime and getting ready for the next morning. Circadian rhythm provides the precise timing mechanism for engaging those different pathways necessary to keep a healthy skin through clock genes that are present in all skin cells. The strongest clue for determining cellular functions timing is through sensing light or absence of light (darkness). Here, we asked the question if blue light could be a direct entrainment signal to skin cells and also disrupt their circadian rhythm at night. Through a reporter assay for per1 transcription, we demonstrate that blue light at 410 nm decreases per1 transcription in keratinocytes, showing that epidermal skin cells can sense light directly and control their own clock gene expression. This triggers cells to "think" it is daytime even at nighttime. Elsewhere, we measured different skin cell damage because of blue light exposure (at different doses and times of exposure) vs. cells that were kept in full darkness. We show an increase in ROS production, DNA damage and inflammatory mediators. These deleterious effects can potentially increase overall skin damage over time and ultimately accelerates ageing.

La peau est exposée chaque jour à de nombreux facteurs de stress et traumatismes environnementaux. Pendant un cycle naturel de 24 heures, dans la journée, la peau est axée sur sa protection, tandis que la nuit, elle se concentre sur la réparation des lésions survenues pendant la journée en préparation du lendemain matin. Le rythme circadien assure un mécanisme de cadencement temporel précis pour engager les différentes voies nécessaires au maintien d'une peau saine à travers les gènes de l'horloge interne qui sont présents dans toutes les cellules cutanées. La perception de la lumière ou l'absence de lumière (obscurité) est le plus fort indice pour déterminer le cadencement des fonctions cellulaires. Nous nous sommes ici posé la question de savoir si la lumière bleue serait un signal d'entraînement direct pour les cellules de la peau également capable de perturber leur rythme circadien la nuit. À travers un essai utilisant un gène rapporteur pour la transcription de per1, nous démontrons que la lumière bleue à 410 nm diminue la transcription de per1 dans les kératinocytes, montrant que les cellules épidermiques peuvent détecter directement la lumière et contrôler l'expression de leurs propres gènes horloges. Cela incite les cellules à « penser ¼ que la journée a commencé, même pendant la nuit. Par ailleurs, nous avons mesuré différentes lésions des cellules cutanées suite à l'exposition à la lumière bleue (à différentes doses et durées d'exposition) par rapport aux cellules qui étaient maintenues dans une obscurité complète. Nous montrons une augmentation de la production d'espèces réactives de l'oxygène (ROS), des lésions de l'ADN et des médiateurs inflammatoires. Ces effets délétères peuvent potentiellement augmenter les lésions cutanées globales au fil du temps et en accélérer ultérieurement le vieillissement.

Effect of SIRT6 knockdown on NF-κB induction and on residual DNA damage in cultured human skin fibroblasts.

SIRT6 is a member of the sirtuin family, which is involved in multiple cellular pathways related to aging, inflammation, epigenetics, and a variety of other cellular functions, including DNA repair (1). Multiple pathways involving different cellular functions are impacted by the deacetylase activity of SIRT6. Genomic integrity is maintained by the capacity of SIRT6 to modulate the accessibility of DNA repair proteins. Glucose metabolism is suppressed by SIRT6 via the deacetylation of histones located at the promoter regions of multiple glycolytic genes and the corepression of hypoxia-inducible factor-1α. SIRT6 is also a corepressor of nuclear factor (NF)-κB, silencing NF-κB target genes through the deacetylation of histones at their promoters' regions. We used SIRT6 small-interfering RNA as a tool to modulate residual DNA damage and NF-κB expression in human dermal fibroblasts. We measured NF-κB levels in the presence or the absence of ultraviolet B (UVB). The impact of SIRT6 knockdown as shown by a decrease in SIRT6 messenger RNA levels resulted in residual DNA damage as evaluated by the comet assay. Our results show that NF-κB was increased significantly (up to 400%) due to SIRT6 silencing in the absence of UVB, illustrating the master regulatory function of SIRT6 in inflammation. We also found a significant increase in DNA damage without UV exposure as a result of SIRT6 silencing, indicating the importance of SIRT6 in DNA repair pathways in cultured human dermal fibroblasts.

Templated biomineralization on self-assembled protein fibers.

Biological mineralization of tissues in living organisms relies on proteins that preferentially nucleate minerals and control their growth. This process is often referred to as "templating," but this term has become generic, denoting various proposed mineral-organic interactions including both chemical and structural affinities. Here, we present an approach using self-assembled networks of elastin and fibronectin fibers, similar to the extracellular matrix. When induced onto negatively charged sulfonated polystyrene surfaces, these proteins form fiber networks of approximately 10-mum spacing, leaving open regions of disorganized protein between them. We introduce an atomic force microscopy-based technique to measure the elastic modulus of both structured and disorganized protein before and during calcium carbonate mineralization. Mineral-induced thickening and stiffening of the protein fibers during early stages of mineralization is clearly demonstrated, well before discrete mineral crystals are large enough to image by atomic force microscopy. Calcium carbonate stiffens the protein fibers selectively without affecting the regions between them, emphasizing interactions between the mineral and the organized protein fibers. Late-stage observations by optical microscopy and secondary ion mass spectroscopy reveal that Ca is concentrated along the protein fibers and that crystals form preferentially on the fiber crossings. We demonstrate that organized versus unstructured proteins can be assembled mere nanometers apart and probed in identical environments, where mineralization is proved to require the structural organization imposed by fibrillogenesis of the extracellular matrix.

Field and pore size dependence of the electrophoretic mobility of DNA: a combination of fluorescence recovery after photobleaching and electric birefringence measurements.

By combining an electrophoretic cell with a setup of fluorescence recovery after photobleaching (FRAP) we can measure the electrophoretic mobility mu of double-stranded lambda DNA in agarose gel as a function of electric field E and gel concentration C. Mobility varies linearly with the field in agreement with the biased reptation model with fluctuations. The slopes are analyzed in term of orientation and compared with birefringence results. The mobility extrapolated at zero field follows the prediction of the reptation theory; we deduced the variation of the pore size with the agarose concentration. With a special use of our setup, we measure directly the free-mobility mu 0 of the DNA.

Agarose gel structure using atomic force microscopy: gel concentration and ionic strength effects.

Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.

Pore size of agarose gels by atomic force microscopy.

The pore size of agarose gel in water at different concentrations was directly measured using atomic force microscopy (AFM). The experiment was specially designed to work under aqueous conditions and allows direct observation of the "unperturbed" gel without invasive treatment. The pore size a as a function of gel concentration C shows a power law dependence a approximately C-gamma, where gamma lies between the prediction of the Ogston model for a random array of straight chains, 0.5, and the value predicted by De Gennes for a network of flexible chains, 0.75. We confirm that gels present a wide pore size distribution and show that it narrows as the concentration increases.

Band broadening in gel electrophoresis: scaling laws for the dispersion coefficient measured by FRAP.

We determined quantitatively the band broadening effect during gel electrophoresis by measuring the longitudinal dispersion coefficient Dx, with a fluorescence recovery after photobleaching setup, coupled to an electrophoretic cell. We carried out measurements as a function of the electric field, the average pore size, and the molecular length of DNA fragments. Our results are in good agreement with the predictions of the biased reptation model with fluctuations described by T. A. Duke et al. [(1992) Physics Review Letters, vol. 69, pp. 3260-3263]. This agreement is observed on single-stranded DNA [persistence length approximately equal to 4 nm; B. Tinland et al. (1997) Macromolecules, vol. 30, pp. 5763-5765] in polyacrylamide gels and on double-stranded DNA (persistence length approximately equal to 50 nm) in agarose gels, two systems where the ratio between the average pore size and the Kuhn length is larger than 1.

DNA electrophoresis on a flat surface.

We report a new approach for performing DNA electrophoresis. Using experimental studies and molecular dynamics simulations, we show that a perfectly flat silicon wafer, without any surface features, can be used to fractionate DNA in free solution. We determine that the ability of a flat surface to separate DNA molecules results from the local friction between the surface and the adsorbed DNA segments. We control this friction by coating the Si surface with silane monolayer films and show that it is possible to systematically change the size range of DNA that can be separated.