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Synthesis of new hydrophilic rhodamine based enzymatic substrates compatible with droplet-based microfluidic assays
Johan Fenneteau, Dany Chauvin,b Andrew D. Griffiths,b Clément Nizak,b and Janine Cossy
Chem. Comm. - 53 5437-5440 - DOI: 10.1039/C7CC01506B - 2017
Here we report the conception, synthesis and evaluation of new hydrophilic rhodamine-based enzymatic substrates for detection of peptidase activity compatible with high-throughput screening using droplet-based microfluidics.
Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica
Beneyton T, Thomas S, Griffiths AD, Nicaud JM, Drevelle A, Rossignol T.
Microb Cell Fact. - 16(1) 18 - doi: 10.1186/s12934-017-0629-5. - 2017
BACKGROUND:
Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.
RESULTS:
Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).
CONCLUSIONS:
Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.
Sign epistasis caused by hierarchy within signalling cascades.
Nghe P, Kogenaru M, Tans SJ.
Nat Commun - 9(1) 1451. - doi: 10.1038/s41467-018-03644-8 - 2017
Sign epistasis is a central evolutionary constraint, but its causal factors remain difficult to predict. Here we use the notion of parameterised optima to explain epistasis within a signalling cascade, and test these predictions in Escherichia coli. We show that sign epistasis arises from the benefit of tuning phenotypic parameters of cascade genes with respect to each other, rather than from their complex and incompletely known genetic bases. Specifically, sign epistasis requires only that the optimal phenotypic parameters of one gene depend on the phenotypic parameters of another, independent of other details, such as activating or repressing nature, position within the cascade, intra-genic pleiotropy or genotype. Mutational effects change sign more readily in downstream genes, indicating that optimising downstream genes is more constrained. The findings show that sign epistasis results from the inherent upstream-downstream hierarchy between signalling cascade genes, and can be addressed without exhaustive genotypic mapping.
Derivation of nearest-neighbor DNA parameters in magnesium from single molecule experiments.
Huguet JM1,2, Ribezzi-Crivellari M3, Bizarro CV4, Ritort F1,5.
Nucleic Acids Res. - 120 158101 - doi: 10.1093/nar/gkx1161. - 2017
DNA hybridization is an essential molecular reaction in biology with many applications. The nearest-neighbor (NN) model for nucleic acids predicts DNA thermodynamics using energy values for the different base pair motifs. These values have been derived from melting experiments in monovalent and divalent salt and applied to predict melting temperatures of oligos within a few degrees. However, an improved determination of the NN energy values and their salt dependencies in magnesium is still needed for current biotechnological applications seeking high selectivity in the hybridization of synthetic DNAs. We developed a methodology based on single molecule unzipping experiments to derive accurate NN energy values and initiation factors for DNA. A new set of values in magnesium is derived, which reproduces unzipping data and improves melting temperature predictions for all available oligo lengths, in a range of temperature and salt conditions where correlation effects between the magnesium bound ions are weak. The NN salt correction parameters are shown to correlate to the GC content of the NN motifs. Our study shows the power of single-molecule force spectroscopy assays to unravel novel features of nucleic acids such as sequence-dependent salt corrections.
Information-theoretic analysis of the directional influence between cellular processes
Sourabh Lahiri, Philippe Nghe, Sander J. Tans, Martin Luc Rosinberg, David Lacoste
Nucleic Acids Res. - - doi.org/10.1371/journal.pone.0187431 - 2017
Inferring the directionality of interactions between cellular processes is a major challenge in systems biology. Time-lagged correlations allow to discriminate between alternative models, but they still rely on assumed underlying interactions. Here, we use the transfer entropy (TE), an information-theoretic quantity that quantifies the directional influence between fluctuating variables in a model-free way. We present a theoretical approach to compute the transfer entropy, even when the noise has an extrinsic component or in the presence of feedback. We re-analyze the experimental data from Kiviet et al. (2014) where fluctuations in gene expression of metabolic enzymes and growth rate have been measured in single cells of E. coli. We confirm the formerly detected modes between growth and gene expression, while prescribing more stringent conditions on the structure of noise sources. We furthermore point out practical requirements in terms of length of time series and sampling time which must be satisfied in order to infer optimally transfer entropy from times series of fluctuations
Evolutionary Applications
Calcagno, V., Mitoyen, C., Audiot, P., Ponsard, S., Gao, G.-Z., Lu, Z.-Z., Wang, Z.-Y., He, K.-L., and Bourguet, D. Parallel
Semin Cell Dev Biol. - 10 9 - DOI: 10.1111/eva.12481 - 2017
Maize was introduced into opposite sides of Eurasia 500 years ago, in Western Europe and in Asia. This caused two host-shifts in the phytophagous genus Ostrinia; O. nubilalis (the European corn borer; ECB) and O. furnacalis (the Asian corn borer; ACB) are now major pests of maize worldwide. They originated independently from Dicot-feeding ancestors, similar to O. scapulalis(the Adzuki bean borer; ABB). Unlike other host-plants, maize is yearly harvested, and harvesting practices impose severe mortality on larvae found above the cut-off line. Positive geotaxis in the ECB has been proposed as a behavioural adaptation to harvesting practices, allowing larvae to move below the cut-off line and thus escape harvest mortality. Here, we test whether the same behavioural adaptation evolved independently in Europe and in Asia. We sampled eight genetically differentiated ECB, ACB and ABB populations in France and China and monitored geotaxis through the entire larval development in artificial stacks mimicking maize stems. We find that all ECB and ACB populations show a similar tendency to move down during the latest larval stages, a behaviour not observed in any European or Asian ABB population. The behaviour is robustly expressed regardless of larval density, development mode or environmental conditions. Our results indicate that maize introduction triggered parallel behavioural adaptations in Europe and Asia, harvest selection presumably being the main driver.
Nanoscale capillary freezing of ionic liquids confined between metallic interfaces and the role of electronic screening
J. Comtet, A. Niguès, V. Kaiser, B. Coasne, L. Bocquet and A. Siria, Nature Materials
Nature Materials - 16 634‐639 - doi: 10.1038/nmat4880 - 2017
Room-temperature ionic liquids (RTILs) are new materials with fundamental importance for energy storage and active lubrication. They are unusual liquids, which challenge the classical frameworks of electrolytes, whose behaviour at electrified interfaces remains elusive, with exotic responses relevant to their electrochemical activity. Using tuning-fork-based atomic force microscope nanorheological measurements, we explore here the properties of confined RTILs, unveiling a dramatic change of the RTIL towards a solid-like phase below a threshold thickness, pointing to capillary freezing in confinement. This threshold is related to the metallic nature of the confining materials, with more metallic surfaces facilitating freezing. This behaviour is interpreted in terms of the shift of the freezing transition, taking into account the influence of the electronic screening on RTIL wetting of the confining surfaces. Our findings provide fresh views on the properties of confined RTIL with implications for their properties inside nanoporous metallic structures, and suggests applications to tune nanoscale lubrication with phase-changing RTILs, by varying the nature and patterning of the substrate, and application of active polarization.
Contact dependence and velocity crossover in friction between microscopic solid/solid contacts
McGraw, A. Niguès, A. Chennevière, A. Siria
Nano Lett. - 17 (10) 6335–6339 - DOI: 10.1021/acs.nanolett.7b03076 - 2017
Friction at the nanoscale differs markedly from that between surfaces of macroscopic extent. Characteristically, the velocity dependence of friction between apparent solid/solid contacts can strongly deviate from the classically assumed velocity independence. Here, we show that a nondestructive friction between solid tips with radius on the scale of hundreds of nanometers and solid hydrophobic self-assembled monolayers has a strong velocity dependence. Specifically, using laterally oscillating quartz tuning forks, we observe a linear scaling in the velocity at the lowest accessed velocities, typically hundreds of micrometers per second, crossing over into a logarithmic velocity dependence. This crossover is consistent with a general multicontact friction model that includes thermally activated breaking of the contacts at subnanometric elongation. We find as well a strong dependence of the friction on the dimensions of the frictional probe.
New avenues for the large-scale harvesting of blue energy
Alessandro Siria, Marie-Laure Bocquet & Lydéric Bocquet
Nature Reviews Chemistry - 91 - doi:10.1038/s41570-017-0091 - 2017
Salinity gradients have been identified as promising clean, renewable and non-intermittent sources of energy — so-called blue energy. However, the low efficiency of current harvesting technologies is a major limitation for large-scale viability and is mostly due to the low performances of the membrane processes currently in use. Advances in materials fabrication with dedicated chemical properties can resolve this bottleneck and lead to a new class of membranes for blue-energy conversion. In this Perspective, we briefly present current technologies for the conversion of blue energy, describe their performances and note their limitations. We then discuss new avenues for the development of a new class of membranes, combining considerations in nanoscale fluid dynamics and surface chemistry. Finally, we discuss how new functionalities originating from the exotic behaviour of fluids in the nanoscale regime can further boost energy conversion, making osmotic energy a tangible, clean alternative.
“ The Landau-Squire plume
Eleonora Secchi, Sophie Marbach, Antoine Niguès, Alessandro Siria
J Phys Chem C - - doi.org/10.1017/jfm.2017.441 - 2017
In this paper, we analyse the dispersion of a dye by a Landau–Squire plume, generated by a jet flow emerging from a nanocapillary into a reservoir. We demonstrate analytically that the dye concentration profile exhibits a long-range profile decaying as the inverse of the distance to the origin, whereas the plume shape is only a function of a Péclet number defined in terms of the flow characteristics inside the nanocapillary. These predictions are successfully compared with experiments on fluorescent dye dispersion from nanocapillaries under pressure-driven flow. The plume shape allows extraction of the nanojet force characterizing the Landau–Squire velocity profile for a given pressure drop, with results in full agreement with direct velocimetry measurements and finite-element calculations. The peculiarities of the Landau–Squire plume make it a sensitive probe of the flow properties inside the seeding nanocapillary.

410 publications.