Sulfopeptide CLEL6 inhibits anthocyanin biosynthesis in Arabidopsis thaliana.

Posttranslationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small posttranslationally modified peptides are perceived by leucine-rich repeat receptor-like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibits anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is downregulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continues to act as negative regulators to limit pigment accumulation in the light.

Bending Actuation of Hydrogels through Rotation of Light-Driven Molecular Motors.

The unidirectional rotation of chemically crosslinked light-driven molecular motors is shown to progressively shift the swelling equilibrium of hydrogels. The concentration of molecular motors and the initial strand density of the polymer network are key parameters to modulate the macroscopic contraction of the material, and both parameters can be tuned using polymer chains of different molecular weights. These findings led to the design of optimized hydrogels revealing a half-time contraction of approximately 5 min. Furthermore, under inhomogeneous stimulation, the local contraction event was exploited to design useful bending actuators with an energy output 400 times higher than for previously reported self-assembled systems involving rotary motors. In the present configuration, we measure that a single molecular motor can lift up loads of 200 times its own molecular weight.

Proteoid biodynamers for safe mRNA transfection via pH-responsive nanorods enabling endosomal escape.

The recent success of mRNA vaccines using lipid-based vectors highlights the importance of strategies for nucleotide delivery under the pandemic situation. Although current mRNA delivery is focused on lipid-based vectors, still they need to be optimized for increasing stability, targeting, and efficiency, and for reducing toxicity. In this regard, other vector systems featuring smart strategies such as pH-responsive degradability and endosomal escape ability hold the potential to overcome the current limitations. Here, we report pH-responsive polymeric nanorods made of amino acid-derivatives connected by dynamic covalent bonds called proteoid-biodynamers, as mRNA vectors. They show excellent biocompatibility due to the biodegradation, and outstanding transfection. The biodynamers of Lys, His, and Arg or monomer mixtures thereof were shown to form nanocomplexes with mRNA. They outperformed conventional transfection agents three times regarding transfection efficacy in three human cell lines, with 82-98% transfection in living cells. Also, we confirmed that the biodynamers disrupted the endosomes up to 10-fold more in number than the conventional vectors. We discuss here their outstanding performance with a thorough analysis of their nanorod structure changes in endosomal microenvironments.

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action.

Phytosulfokine (PSK) precursor processing by subtilase SBT3.8 and PSK signaling improve drought stress tolerance in Arabidopsis.

Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.

Self-Induced Crystallization in Charged Gold Nanoparticle-Semiflexible Biopolyelectrolyte Complexes.

Mixing negatively charged polyelectrolyte (PEL) with positively charged gold nanoparticles (Au NPs) in aqueous solution results in electrostatics complexes of different shapes and compactness. Here, when complexing with a semirigid PEL hyaluronic acid (HA), we obtain crystals made of nanoparticles in a new region of the phase diagram, as evidenced by small-angle X-ray scattering (SAXS). The Au NPs were initially well dispersed in solution; their size distribution is well controlled but does not need to be extremely narrow. The bacterial hyaluronic acid, polydispersed, is commercially available. Such rather simple materials and mixing preparation produce a highly ordered crystalline phase of electrostatic complexes. The details of the interactions between spherical nanoparticles and linear polymer chains remain to be investigated. In practice, it opens a completely new and unexpected method of complexation. It has high potential, in particular because one can take advantage of the versatility of Au NPs associated with the specificity of biopolymers, varied due to natural biodiversity.

Structural properties of contractile gels based on light-driven molecular motors: a small-angle neutron and X-ray study.

The detailed structure of active polymer gels built by integrating light-driven rotary molecular motors as reticulation units in polymer networks is discussed as a function of gel composition. Upon light-irradiation, the collective rotation of molecular motors is translated into the macroscopic contraction of the gels through polymer chains twisting. The major role of the characteristic ratio c/c* (c* being the overlap concentration of the polymer-motor conjugates before crosslinking) on the contraction efficiency is exploited. Combined small-angle neutron and X-ray scattering experiments reveal the importance of heterogeneities in the macroscopic contraction process: the mesh size of the network increases under irradiation in the whole range of c/c*, an increase that is maximal for c/c* = 1; i.e. at higher contraction efficiency. Furthermore, the mesh size of the network reaches equilibrium within a short period of time, while the heterogeneities increase in size untill the end of the contraction process. Finally, the significant motorized twisting of polymer chains within the network allows to foresee the design of new storage energy systems.

Homodyne dynamic light scattering in supramolecular polymer solutions: anomalous oscillations in intensity correlation function.

Dilute solutions of electronically active molecules capable of irradiation-driven supramolecular self-assembly are studied by dynamic light scattering. We detect unusual well-defined oscillations in the long time range of the homodyne intensity correlation function for all solutions that were irradiated with white light prior to the measurements. The oscillation effect is attributed to the local laser-induced heating of the samples due to strongly enhanced absorption manifested by the supramolecular filaments. It is found that the oscillation frequency depends on the irradiation time, solution concentration, and the incident laser power, but is independent of the scattering angle. These observations are explained with a semi-quantitative theory relating the oscillation effect to thermo-gravitational convection flows generated by laser beam. The results suggest that the presence of such homodyne oscillations could be a sensitive probe for aggregation in many complex systems.

Mechanical behaviour of contractile gels based on light-driven molecular motors.

The networking of individual artificial molecular motors into collective actuation systems is a promising approach for the design of active materials working out of thermodynamic equilibrium. Here, we report the first mechanical studies on active polymer gels built by integrating light-driven rotary molecular motors as reticulation units in polymer networks. We correlate the volume ratio before and after light irradiation with the change of the elastic modulus, and we reveal the universal maximum mechanical efficiency of such gels related to their critical overlap concentration before chemical reticulation. We also show the major importance of heterogeneities in the macroscopic contraction process and we confirm that these materials can increase their internal energy by the motorized winding of their polymer chains.

Autopoietic Behavior of Dynamic Covalent Amphiphiles.

The condensation of aldehydes and amines in water to give amphiphilic imines can lead to a particular autocatalytic behavior known as autopoiesis, in which the closed micellar structure made by the amphiphile at the mesoscale can accelerate the condensation of its constituents. Herein, through a combination of analytical tools, including diffusion ordered spectroscopy (DOSY) as well as light, neutron, and X-ray scattering techniques, the thermodynamic and kinetic parameters were probed at both the level of dynamic covalent imine bond formation and the level of the resulting micellar self-assemblies. It was found that the autopoietic behavior was the result of a combination of several parameters, including solubilization of hydrophobic building blocks, template effect at the core-shell interface, and growth/division cycles of the micellar objects.

3D supramolecular self-assembly of [60]fullerene hexaadducts decorated with triarylamine molecules.

A clickable fullerene hexa-adduct scaffold has been functionalized with twelve triarylamine subunits. The light-triggered self-assembly of this molecular unit leads to 3D honeycomb-like structures with inner pores of around 10 nm diameter. Multiple grafting of triarylamine subunits onto a hard-core C60 unit increases the dimensionality of the self-assembly process by reticulating the 1D nanowires typically obtained from the supramolecular polymerization of triarylamine monomers.

Dynamic Proteoids Generated From Dipeptide-Based Monomers.

Dynamic proteoids are dynamic covalent analogues of proteins which are generated through the reversible polymerization of amino-acid- or peptide-derived monomers. The authors design and prepare a series of dynamic proteoids based on the reversible polycondensation of six types of dipeptide hydrazides bearing different categories of side chains. The polymerization and structures of biodynamers generated by 1 H-NMR spectroscopy, light scattering and cryo-transmission-electron microscopy are studied. This study shows that the presence of aromatic rings in the side chains plays the most essential role in determining the extent of the polymerization and organization into resultant nanostructures through π-π-stacking interactions, hydroxyl groups have a less favorable influence via hydrogen bonds, whereas a high density of positive charge blocks the generation of biodynamers due to electrostatic repulsions. These findings set the stage for the rational design and synthesis of dynamic proteoids as novel biofunctional materials.

Lipid-DNAs as Solubilizers of mTHPC.

Hydrophobic drug candidates require innovative formulation agents. We designed and synthesized lipid-DNA polymers containing varying numbers of hydrophobic alkyl chains. The hydrophobicity of these amphiphiles is easily tunable by introducing a defined number of alkyl chain-modified nucleotides during standard solid-phase synthesis of DNA using an automated DNA synthesizer. We observed that the resulting self-assembled micelles solubilize the poorly water-soluble drug, meta-tetra-hydroxyphenyl-chlorin (mTHPC) used in photodynamic therapy (PDT) with high loading concentrations and loading capacities. A cell viability study showed that mTHPC-loaded micelles exhibit good biocompatibility without irradiation, and high PDT efficacy upon irradiation. Lipid-DNAs provide a novel class of drug-delivery vehicle, and hybridization of DNA offers a potentially facile route for further functionalization of the drug-delivery system with, for instance, targeting or imaging moieties.

Integration of molecular machines into supramolecular materials: actuation between equilibrium polymers and crystal-like gels.

In this article, the dynamic structure of complex supramolecular polymers composed of bistable [c2]daisy chain rotaxanes as molecular machines that are linked by ureidopyrimidinones (Upy) as recognition moieties was studied. pH actuation of the integrated mechanically active rotaxanes controls the contraction/extension of the polymer chains as well as their physical reticulation. Small-angle neutron and X-ray scattering were used to study in-depth the nanostructure of the contracted and extended polymer aggregates in toluene solution. The supramolecular polymers comprising contracted nanomachines were found to be equilibrium polymers with a mass that is concentration dependent in dilute and semidilute regimes. Surprisingly, the extended polymers form a gel network with a crystal-like internal structure that is independent of concentration and reminiscent of a pearl-necklace network.

Saccharide-Containing Dynamic Proteoids.

Dynamic proteoids are dynamic covalent analogues of proteins, which can be used as new adaptive biomaterials. We designed and synthesized a range of sugar-containing dynamic proteoid biodynamers based on the polycondensation of different types of amino acid and dipeptide hydrazides with a biological aliphatic dialdehyde and a nonbiological aromatic dialdehyde. By using the saccharide-based dialdehyde, the biocompatibility of biodynamers should be enhanced compared to previously reported biodynamers.

Bistable [c2] Daisy Chain Rotaxanes as Reversible Muscle-like Actuators in Mechanically Active Gels.

The implementation of molecular machines in polymer science is of high interest to transfer mechanical motions from nanoscale to macroscale in order to access new kinds of active devices and materials. Toward this objective, thermodynamic and topological aspects need to be explored for reaching efficient systems capable of producing a useful work. In this paper we describe the branched polymerization of pH-sensitive bistable [c2] daisy chain rotaxanes by using copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition ("click chemistry"). With this cross-linked topology, the corresponding materials in the form of chemical gels can be contracted and expanded over a large variation of volume (∼50%) by changing the protonation state of the system. HR-MAS 1H NMR and neutron scattering experiments reveal that this macroscopic response of the gels results from the synchronized actuation of the mechanical bonds at the molecular level.

Controlled Sol-Gel Transitions by Actuating Molecular Machine Based Supramolecular Polymers.

The implementation of artificial molecular machines in polymer science is an important objective that challenges chemists and physicists in order to access an entirely new class of smart materials. To design such systems, the amplification of a mechanical actuation from the nanoscale up to a macroscopic response in the bulk material is a central issue. In this article we show that bistable [c2]daisy chain rotaxanes (i.e., molecular muscles) can be linked into main-chain Upy-based supramolecular polymers. We then reveal by an in depth quantitative study that the pH actuation of the mechanically active rotaxane at the nanoscale influences the physical reticulation of the polymer chains by changing the supramolecular behavior of the Upy units. This nanoactuation within the local structure of the main chain polymer results in a mechanically controlled sol-gel transition at the macroscopic level.

How does the size of gold nanoparticles depend on citrate to gold ratio in Turkevich synthesis? Final answer to a debated question.

The dependence between the size of gold nanoparticle (AuNP) and the citrate to gold molar ratio (X) is still a matter of debate 65years after the seminal work by Turkevich et al. for high X values. We assume that this dispersion of results is due to the variation of certain parameters that are often not mentioned in the protocols, and to the use of a single characterization technique (dynamic light scattering (DLS) or transmission electron microscopy (TEM)). To adress definitely the question of this dependence, we have synthesized AuNPs with very precise protocols ensuring that the only parameters to be modified are X and the sequence of reagent addition. We have then studied, for the first time, the dependence of the size with X quantitatively with a multimodal approach (UV-Visible spectroscopy, DLS and TEM) for 2 synthetic routes differing only by the sequence of reagent addition. We show unambiguously that AuNPs' size decay monotonically, with X whatever the order of reagent addition. It allows us to exclude the occurrence of a measurable discontinuity for a peculiar value of X that prompted some authors to postulate the existence of two different reaction pathways when the citrate to gold molar ratio is around this value. In contrast, our result is in line with one reaction pathway, likely a "seed-mediated" growth mechanism, which should leads to monotonic size decrease. Also, we note that our result agrees with the sole available theoretical prediction (Kumar et al., 2007) on the whole range of X. Despite this apparent agreement, we point some contradictions between recent experimental results and basal hypothesis of this model.

Role of the ratio of biopolyelectrolyte persistence length to nanoparticle size in the structural tuning of electrostatic complexes.

Aggregation of nanoparticles of given size R induced by addition of a polymer strongly depends on its degree of rigidity. This is shown here on a large variety of silica nanoparticle self-assemblies obtained by electrostatic complexation with carefully selected oppositely charged biopolyelectrolytes of different rigidity. The effective rigidity is quantified by the total persistence length L_{T} representing the sum of the intrinsic (L_{p}) and electrostatic (L_{e}) polyelectrolyte persistence length, which depends on the screening, i.e., on ionic strength due to counterions and external salt concentrations. We experimentally show that the ratio L_{T}/R is the main tuning parameter that controls the fractal dimension D_{f} of the nanoparticles' self-assemblies, which is determined using small-angle neutron scattering: (i) For L_{T}/R<0.3 (obtained with flexible poly-l-lysine in the presence of an excess of salt), chain flexibility promotes easy wrapping around nanoparticles in excess, hence ramified structures with D_{f}∼2. (ii) For 0.31,L_{e} is strongly increased due to the absence of salt and repulsions between nanoparticles cannot be compensated for by the polyelectrolyte wrapping, which allows a spacing between nanoparticles and the formation of one-dimensional pearl necklace complexes. (iv) Finally, electrostatic screening, i.e., ionic strength, turned out to be a reliable way of controlling D_{f} and the phase diagram behavior. It finely tunes the short-range interparticle potential, resulting in larger fractal dimensions at higher ionic strength.

An Easily Accessible Self-Healing Transparent Film Based on a 2D Supramolecular Network of Hydrogen-Bonding Interactions between Polymeric Chains.

Self-healing polymers hold great promise for the future, enhancing in particular the longevity of polymeric materials. We describe a self-healing covalent polymer, presenting an extensive array of hydrogen-bonding sites based on the combination of urea, urethane, and bis-acyl-hydrazine units. Solvent-cast thin-films prepared by polycondensation of a commercially available dihydrazide and a diisocyanate prepolymer exhibited excellent room temperature autonomous healing with almost full recovery of mechanical properties when two parts of a cut film were overlapped and gently pressed together. This autonomous healing upon damage may be attributed to the supramolecular dynamics of multiple lateral inter-chain hydrogen-bonding interactions between the polymer chains. The solid-state structure of a model compound incorporating the same structural backbone corroborates the existence of an extensive two-dimensional supramolecular hydrogen-bonding network.