Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity.

Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5' monophosphate and a 3' stable hairpin is highly susceptible to poly(A)-dependent degradation.

Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is
becoming a widespread method for performing assays and especially in the cell biol-
ogy field. Making a simple and highly portable system for creating emulsion droplets
would help to continue the popularization of such a technique. Also, the ability to
emulsify all the samples would strengthen this compartimenlization technique to han-
dle samples with limited volume. Here, we propose a strategy of droplet formation
that combines a classical flow-focusing microfluidic chip, which could be commer-
cially available, with a standard laboratory adjustable micropipette. The micropipette
is used as a negative pressure generator for controlling liquid flows. In that way, emul-
sification does neither require any electrical power supply nor a cumbersome device
and functions with small liquid volumes. Droplet formation can be easily and safely
performed in places with limited space, opening a wide range of applications espe-
cially in biological laboratory environments with higher level of safety regulations,
i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid vol-
umes, and thus possible time dependent flow conditions, allows to minimize dead
volume while keeping drops’ size homogeneous. A physical characterization
of droplet production and a model that describes the emulsion features, in terms of
drop size and size distribution, are proposed for rationalizing the performances of
the micropipette-powered emulsification process.
Published by AIP Publishing.

Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Copyright © 2018 Elsevier B.V. All rights reserved.

Aluminium salts are widely used to control sweating for personal hygiene purposes. Their mechanism of action as antiperspirants was previously thought to be a superficial plugging of eccrine sweat pores by the aluminium hydroxide gel. Here we present a microfluidic T junction device that mimics sweat ducts, and is designed for the real time study of interactions between sweat and ACH (Aluminium Chloro Hydrate) under conditions that lead to plug formation. We used this device to image and measure the diffusion of aluminium polycationic species in sweat counter flow. We report the results of small angle X-ray scattering experiments performed to determine the structure and composition of the plug, using BSA (Bovine Serum Albumin) as a model of sweat proteins. Our results show that pore occlusion occurs as a result of the aggregation of sweat proteins by aluminium polycations. Mapping of the device shows that this aggregation is initiated in the T junction at the location where the flow of aluminium polycations joins the flow of BSA. The mechanism involves two stages: (1) a nucleation stage in which aggregates of protein and polycations bind to the wall of the sweat duct and form a tenuous membrane, which extends across the junction; (2) a growth stage in which this membrane collects proteins that are carried by hydrodynamic flow in the sweat channel and polycations that diffuse into this channel. These results could open up perspectives to find new antiperspirant agents with an improved efficacy.

We obtained osmotic pressure data of lysozyme solutions, describing their physical states over a wide concentration range, using osmotic stress for pressures between 0.05 bar and about 40 bar and volume fractions between 0.01 and 0.61. The osmotic pressure vs. volume fraction data consist of a dilute, gas-phase regime, a transition regime with a high-compressibility plateau, and a concentrated regime where the system is nearly incompressible. The first two regimes are shifted towards a higher protein volume fraction upon decreasing the strength or the range of electrostatic interactions. We describe this shift and the overall shape of the experimental data in these two regimes through a model accounting for a steric repulsion, a short-range van der Waals attraction and a screened electrostatic repulsion. The transition is caused by crystallization, as shown by small-angle X-ray scattering. We verified that our data points correspond to thermodynamic equilibria, and thus that they consist of the reference experimental counterpart of a thermodynamic equation of state.

Monte Carlo simulations and coarse-grained modeling have been used to analyze Histatin 5, an unstructured short cationic salivary peptide known to have anticandidical properties. The calculated scattering functions have been compared with intensity curves and the distance distribution function P(r) obtained from small angle X-ray scattering (SAXS), at both high and low salt concentrations. The aim was to achieve a molecular understanding and a physico-chemical insight of the obtained SAXS results and to gain information of the conformational changes of Histatin 5 due to altering salt content, charge distribution, and net charge. From a modeling perspective, the accuracy of the electrostatic interactions are of special interest. The used coarse-grained model was based on the primitive model in which charged hard spheres differing in charge and in size represent the ionic particles, and the solvent only enters the model through its relative permittivity. The Hamiltonian of the model comprises three different contributions: (i) excluded volumes, (ii) electrostatic, and (iii) van der Waals interactions. Even though the model can be considered as gross omitting all atomistic details, a great correspondence is obtained with the experimental results. Proteins 2016; 84:777-791.

Liquid jets ultimately break up into droplets through an instability driven by surface tension. For highly viscous liquids, drops are connected by cylindrical liquid filaments whose radii linearly decrease with time, thus forming drops on a string structure. For a jet composed of two aqueous phases made in air by coaxial extrusion, we observe that, for moderate Weber and capillary numbers, drops slow down with different velocities, leading to drop coalescence. The origin of the traffic collision is linked to the spatial feature of the capillary instability where capillary and viscous forces acting on the drops evolve along the jet and ultimately amplify small velocity fluctuations. The emergence of such fluctuations is related to the unstable nature of the annular coflow of liquids having contrasting viscoelastic properties. From a practical point of view, flow and actuation conditions can be adjusted to inhibit drop collision and thus drop coalescence. These findings allow then the fabrication of monodisperse submillimeter core-shell objects based on the fragmentation of compound jets made of polymer solutions that find applications for three-dimensional cell culture.

The fabrication of alginate hydrogel microparticles with embedded liposomes and magnetic nanoparticles for radiofrequency controlled release of encapsulated chemical cargo was considered. An extractive gelation process was implemented in a microfluidic device, which enabled the production of uniform composite microparticles of dimensions comparable to those of blood cells (between 5 and 10 μm). The critical parameters that control the extractive gelation process were systematically explored and feasible values that provide microgel particles of a defined size and morphology were identified. First, the initial water-in-oil droplet is formed in a flow-focusing junction whose size is controlled by the flow-rate of the oil phase. Then, the train of droplets is sandwiched between two streams of oil containing calcium ions. In that way, a flux of water molecules from the droplets towards the continuous phase as well as a transport of calcium ions towards the disperse phase are initiated. The final microparticle properties were thus found to be sensitive to three elementary sub-processes: (i) the initial droplet size; (ii) the extraction of water into the oil phase, which was controlled by the volume of the oil phase and its initial moisture content; and (iii) the kinetics of ionic cross-linking of the alginate matrix, which was controlled by the varying calcium concentration. The size and morphology of the final composite microgels were fully characterized.

We describe an interfacial rheometry technique based on pairs of micrometer-sized magnetic particles at a fluid–fluid interface. The particles are repeatedly attracted and repelled by well-controlled magnetic dipole–dipole forces, so-called interfacial rheometry by intra-pair magnetophoresis (IPM). From the forces (∼pN), displacements (∼μm) and velocities (∼μm s−1) of the particles we are able to quantify the interfacial drag coefficient of particles within a few seconds and over very long timescales. The use of local dipole–dipole forces makes the system insensitive to fluid flow and suited for simultaneously recording many particles in parallel over a long period of time. We apply IPM to study the time-dependent adsorption of an oil-soluble amino-modified silicone polymer at a water–oil interface using carboxylated magnetic particles. At low polymer concentration the carboxylated particles remain on the water side of the water–oil interface, while at high polymer concentrations the particles transit into the oil phase. Both conditions show a drag coefficient that does not depend on time. However, at intermediate polymer concentrations data show an increase of the interfacial drag coefficient as a function of time, with an increase over more than three orders of magnitude (10−7 to 10−4 N s m−1), pointing to a strong polymer-polymer interaction at the interface. The time-dependence of the interfacial drag appears to be highly sensitive to the polymer concentration and to the ionic strength of the aqueous phase. We foresee that IPM will be a very convenient technique to study fluid–fluid interfaces for a broad range of materials systems.

A modified version of the Gibbs-ensemble Monte-Carlo method reveals how polydisperse charged colloidal particles can build complex colloidal crystals. It provides general rules that are applicable to this fractionated crystallization that stems from size segregation. It explains the spontaneous formation of complex crystals with very large unit-cells in suspensions of nanoparticles with a broad size distribution.

We report small-angle x-ray scattering experiments on aqueous dispersions of colloidal silica with a broad monomodal size distribution (polydispersity, 14%; size, 8 nm). Over a range of volume fractions, the silica particles segregate to build first one, then two distinct sets of colloidal crystals. These dispersions thus demonstrate fractional crystallization and multiple-phase (bcc, Laves AB_{2}, liquid) coexistence. Their remarkable ability to build complex crystal structures from a polydisperse population originates from the intermediate-range nature of interparticle forces, and it suggests routes for designing self-assembling colloidal crystals from the bottom up.

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.

The dissolution and swelling properties of montmorillonite at different pH have been studied, using small angle X-ray scattering (SAXS), imaging and osmotic stress methods combined with Monte Carlo simulations. The acidity of montmorillonite dispersions has been varied as well as the counterions to the net negatively charged platelets. At low pH, Na montmorillonite dissolves and among other species Al3+ is released, hydrated, polymerized and then it replaces the counterions in the clay. This dramatically changes the microstructure of Na montmorillonite, which instead of having fully exfoliated platelets, turns into a structure of aggregated platelets, so-called tactoids. Montmorillonite dispersion still has a significant extra-lamellar swelling among the tactoids due to the presence of very small nanoplatelets.

We present the principle of a fast magnetic field enhanced colloidal agglutination assay, which is based on the acceleration of the recognition rate between ligands and receptors induced by magnetic forces.1 By applying a homogeneous magnetic field of 20 mT for only 7 s, we detect CRP (C-reactive protein) in human serum at a concentration as low as 1 pM for a total cycle time of about 1 min in a prototype analyzer. Such a short measurement time does not impair the performances of the assay when compared to longer experiments. The concentration range dynamic is shown to cover 3 orders of magnitude. An analytical model of agglutination is also successfully fitting our data obtained with a short magnetic pulse.

Aluminium salts are widely used to control sweating for personal hygiene purposes. Their mechanism of action as antiperspirants was previously thought to be a superficial plugging of eccrine sweat pores by the aluminium hydroxide gel. Here we present a microfluidic T junction device that mimics sweat ducts, and is designed for the real time study of interactions between sweat and ACH (Aluminium Chloro Hydrate) under conditions that lead to plug formation. We used this device to image and measure the diffusion of aluminium polycationic species in sweat counter flow. We report the results of small angle X-ray scattering experiments performed to determine the structure and composition of the plug, using BSA (Bovine Serum Albumin) as a model of sweat proteins. Our results show that pore occlusion occurs as a result of the aggregation of sweat proteins by aluminium polycations. Mapping of the device shows that this aggregation is initiated in the T junction at the location where the flow of aluminium polycations joins the flow of BSA. The mechanism involves two stages: (1) a nucleation stage in which aggregates of protein and polycations bind to the wall of the sweat duct and form a tenuous membrane, which extends across the junction; (2) a growth stage in which this membrane collects proteins that are carried by hydrodynamic flow in the sweat channel and polycations that diffuse into this channel. These results could open up perspectives to find new antiperspirant agents with an improved efficacy.