Caged Dexamethasone to Photo-control the Development of Embryos through Activation of the Glucocorticoid Receptor.

Efficient tools for controlling molecular functions with exquisite spatiotemporal resolution are much in demand to investigate biological processes in living systems. Here we report an easily synthesized caged dexamethasone for photo-activating cytoplasmic proteins fused to the glucocorticoid receptor. In the dark, it is stable in vitro as well as in vivo in both zebrafish (Danio rerio) and Xenopus sp, two significant models of vertebrates.

Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain.

The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork.

The Development and Application of Opto-Chemical Tools in the Zebrafish.

The zebrafish is one of the most widely adopted animal models in both basic and translational research. This popularity of the zebrafish results from several advantages such as a high degree of similarity to the human genome, the ease of genetic and chemical perturbations, external fertilization with high fecundity, transparent and fast-developing embryos, and relatively low cost-effective maintenance. In particular, body translucency is a unique feature of zebrafish that is not adequately obtained with other vertebrate organisms.

Vertebrate Cell Differentiation, Evolution, and Diseases: The Vertebrate-Specific Developmental Potential Guardians / and / Enter the Stage.

During vertebrate development, embryonic cells pass through a of transitory pluripotent states that precede multi-lineage commitment and morphogenesis. Such states are referred to as "refractory/naïve" and "competent/formative" pluripotency. The molecular mechanisms maintaining refractory pluripotency or driving the transition to competent pluripotency, as well as the cues regulating multi-lineage commitment, are evolutionarily conserved.

Fgf8 dynamics and critical slowing down may account for the temperature independence of somitogenesis.

Somitogenesis, the segmentation of the antero-posterior axis in vertebrates, is thought to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. The segment (somite) size is set by the product of the oscillator period and the velocity of the determination wavefront. Surprisingly, while the segmentation period can vary by a factor three between 20 °C and 32 °C, the somite size is constant.

Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA.

Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA.

Optical control of protein activity and gene expression by photoactivation of caged cyclofen.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism.

Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesis.

Surface colonization underpins microbial ecology on terrestrial environments. Although factors that mediate bacteria-substrate adhesion have been extensively studied, their spatiotemporal dynamics during the establishment of microcolonies remains largely unexplored. Here, we use laser ablation and force microscopy to monitor single-cell adhesion during the course of microcolony formation.

Control of Protein Activity and Gene Expression by Cyclofen-OH Uncaging.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism.

Optical Control of Tumor Induction in the Zebrafish.

The zebrafish has become an increasingly popular and valuable cancer model over the past few decades. While most zebrafish cancer models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression in live zebrafish. We utilize this technique to transiently or constitutively activate a typical human oncogene, kRASG12V, in zebrafish embryos and investigate the developmental and tumorigenic phenotypes.

A mechanistic study of helicases with magnetic traps.

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue.

Single molecule studies of helicases with magnetic tweezers.

Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA).

Radiation Modeling and Finite Cloud Effects for Atmospheric Dispersion Calculations in Near-field Applications: Modeling of the Full Scale RDD Experiments with Operational Models in Canada, Part II.

Three radiological dispersal devices were detonated in 2012 under controlled conditions at Defence Research and Development Canada's Experimental Proving Grounds in Suffield, Alberta. Each device comprised a 35-GBq source of (140)La. The dataset obtained is used in this study to assess the MLCD, ADDAM, and RIMPUFF atmospheric dispersion models.

The Sensitivity of Atmospheric Dispersion Calculations in Near-field Applications: Modeling of the Full Scale RDD Experiments with Operational Models in Canada, Part I.

Three radiological dispersal devices were detonated in 2012 under controlled conditions at Defence Research and Development Canada's Experimental Proving Grounds in Suffield, Alberta. Each device comprised a 35-GBq source of (140)La. The dataset obtained is used in this study to assess the MLCD, ADDAM, and RIMPUFF atmospheric dispersion models.

Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity.

How to control proteins with light in living systems.

The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution.

Cell-cell contacts confine public goods diffusion inside Pseudomonas aeruginosa clonal microcolonies.

The maintenance of cooperation in populations where public goods are equally accessible to all but inflict a fitness cost on individual producers is a long-standing puzzle of evolutionary biology. An example of such a scenario is the secretion of siderophores by bacteria into their environment to fetch soluble iron. In a planktonic culture, these molecules diffuse rapidly, such that the same concentration is experienced by all bacteria.

Optical control and study of biological processes at the single-cell level in a live organism.

Living organisms are made of cells that are capable of responding to external signals by modifying their internal state and subsequently their external environment. Revealing and understanding the spatio-temporal dynamics of these complex interaction networks is the subject of a field known as systems biology. To investigate these interactions (a necessary step before understanding or modelling them) one needs to develop means to control or interfere spatially and temporally with these processes and to monitor their response on a fast timescale (< minute) and with single-cell resolution.

A blue-absorbing photolabile protecting group for in vivo chromatically orthogonal photoactivation.

The small and synthetically easily accessible 7-diethylamino-4-thiocoumarinylmethyl photolabile protecting group has been validated for uncaging with blue light. It exhibits a significant action cross-section for uncaging in the 470-500 nm wavelength range and a low light absorption between 350 and 400 nm. These attractive features have been implemented in living zebrafish embryos to perform chromatic orthogonal photoactivation of two biologically active species controlling biological development with UV and blue-cyan light sources, respectively.

Validation of a clinical evaluation score for irritative dermatitis: SCOREPI.

Background: Most dermatological conditions can be evaluated using validate clinical scores, no such tool is available for irritant contact dermatitis (ICD).

Objective: To create and validate a grid-based ICD severity score.

Methods: Three dermatologists developed the SCOre de REparation de l'EPIderme (SCOREPI) grid.

Spatiotemporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization.

All-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space.

Monitoring microbial population dynamics at low densities.

We propose a new and simple method for the measurement of microbial concentrations in highly diluted cultures. This method is based on an analysis of the intensity fluctuations of light scattered by microbial cells under laser illumination. Two possible measurement strategies are identified and compared using simulations and measurements of the concentration of gold nanoparticles.

Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient.

Fluorescein monoglycosides (D-galactopyranoside (FMG) and D-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated β-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis.