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Proteinase 3 (PR3), together with other serine proteases, such as neutrophil elastase (NE) and cathepsin G (CG), regulates inflammatory and immune responses. However, in comparison with NE and CG, there is increasing evidence that PR3 functions significantly differ. In particular, PR3 can bind to cell membranes and such membrane-bound PR3 (mbPR3) might be differently involved in the activation of cytokines, growth factors, cellular receptors, and in the regulation of cell apoptosis. For instance, PR3 membrane binding can block some “eat me” signals, notably, phosphatidylserine membrane lipid, and facilitate non-resolving inflammation. Based on the clear evidence that PR3 membrane binding affects the biological functions of PR3, we designed peptidomimetic inhibitors that can remove mbPR3 from the membrane surface in vitro without influencing PR3 catalytic activity. Such inhibitors, which specifically target PR3 binding to membranes, are still lacking. In particular, we found peptidomimetics that inhibit binding of PR3 to POPC:PS liposomes, which mimic the biological environment of PR3. publishedVersion

Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200–400 g/L of macromolecular crowders such as proteins, ribosomes and lipids. Thus we expanded the ITC application for solutions mimicking the cellular environment by adding various macromolecular crowders. We investigated how these crowders affect the kinetics of trypsin-catalyzed reactions and determined the Michaelis-Menten parameters for hydrolysis of two trypsin substrates: Nα-benzoyl- l -arginine ethyl ester (BAEE) and Nα-benzoyl- dl -arginine β-naphthylamide (BANA). Since ITC enables investigations of complex and turbid solutions with label-free reagents, it seems a perfect technique for kinetic analyses in crowded solutions. ITC also offers the opportunity to control enzyme-crowder and substrate-crowder interactions.

International audience; Electrochemical energy converters based on biomass-based organic molecules such as glucose have recently demonstrated great potential for the development of functional bioimplantable devices such as pacemakers, but such converters lack efficient metal-based anode nanocatalysts, which could combine the stability of parameters with high performance. Here, we elaborate a technique of femtosecond laser ablation in water to synthesize bare (ligand-free) PdAu alloy nanoparticles with constituents having a variable ratio and explore these nanoparticles as electrocatalysts for glucose oxidation. Our data evidence that the combination of two metals in one bare nanoformulation can result in a strong synergetic effect, which makes the combination of high catalytic activity toward glucose oxidation with ultrafast kinetics at low potentials possible due to the presence of Au and Pd, respectively, while a bare uncontaminated surface ensures outstanding catalytic performance. We show that at an optimal ratio of constituents (50%/ 50%), PdAu nanoparticles can provide a mass activity of 60 A g −1 and an onset potential lower than 0.27 V vs. RHE, which outperform all currently existing nanoparticle-based electrodes. Our results show that bare nanomaterials prepared by laser-ablation synthesis can offer much improved characteristics as electrocatalysts for energy conversion and storage applications, while the versatility of laser synthesis can be extended to other alloy architectures in order to provide optimal characteristics for a concrete catalytic process.

The widespread emergence of bacterial resistance to existing antibiotics forces the development of new therapeutic agents. The use of short modified oligonucleotides, such as peptide nucleic acids (PNAs), seems a promising strategy. However, the uptake of such oligonucleotides is limited by the bacterial cell wall and is species-dependent. Therefore, new carriers for PNAs should be extensively explored. In this study, we examined the antibacterial activity of vitamin B12–PNA conjugates. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of an essential acpP gene encoding acyl carrier protein in Escherichia coli. PNA–vitamin B12 conjugates were synthesized using the Cu(I)-catalyzed 1,3-dipolar cycloaddition. We examined two types of linkers between vitamin B12 and PNA, including a cleavable disulfide bond. As a positive control for PNA uptake, we used PNA conjugated to the most widely used cell-penetrating peptide (KFF)3K. We found that vitamin B12–PNA conjugates inhibit E. coli growt...

Gram-negative bacteria develop specific systems for the uptake of scarce nutrients, including vitamin B12 . These uptake pathways may be utilized for the delivery of biologically relevant molecules into cells. Indeed, it was recently reported that vitamin B12 transported an antisense peptide nucleic acid (PNA) into Escherichia coli and Salmonella Typhimurium cells. The present studies indicate that the conjugation site of PNA to vitamin B12 has an impact on PNA transport into bacterial cells. Toward this end, a specifically designed PNA oligomer has been tethered at various positions of vitamin B12 (central Co, R5' -OH, c and e amide chains, meso position, and at the hydroxy group of cobinamide) by using known or newly developed methodologies and tested for the uptake of the synthesized conjugates by E. coli. Compounds in which the PNA oligonucleotide was anchored at the R5' -OH position were transported more efficiently than that of other compounds tethered at the peripheral positions around the corrin ring. Of importance is the fact that, contrary to mammalian organisms, E. coli also takes up cobinamide, which is an incomplete corrinoid. This selectivity opens up ways to fight bacterial infections. Gram-negative bacteria develop specific systems for the uptake of scarce nutrients, including vitamin B12 . These uptake pathways may be utilized for the delivery of biologically relevant molecules into cells. Indeed, it was recently reported that vitamin B12 transported an antisense peptide nucleic acid (PNA) into Escherichia coli and Salmonella Typhimurium cells. The present studies indicate that the conjugation site of PNA to vitamin B12 has an impact on PNA transport into bacterial cells. Toward this end, a specifically designed PNA oligomer has been tethered at various positions of vitamin B12 (central Co, R5' -OH, c and e amide chains, meso position, and at the hydroxy group of cobinamide) by using known or newly developed methodologies and tested for the uptake of the synthesized conjugates by E. coli. Compounds in which the PNA oligonucleotide was anchored at the R5' -OH position were transported more efficiently than that of other compounds tethered at the peripheral positions around the corrin ring. Of importance is the fact that, contrary to mammalian organisms, E. coli also takes up cobinamide, which is an incomplete corrinoid. This selectivity opens up ways to fight bacterial infections.

Cells are crowded with various macromolecules and metabolites, which affect biochemical reactions in many ways, from the diffusion of substrates to catalytic activities of enzymes. We herein investigated the proteolytic activity of the human immunodeficiency virus type 1 protease (HIV-1 PR) under non-crowded and crowded conditions. The latter environment was mimicked with various (poly)ethylene glycol molecules as crowding agents. We found that these crowding agents affect the kinetic parameters of the HIV-1 PR catalyzed reaction by increasing the Michaelis–Menten constant and decreasing the maximum velocity. The influence of crowding was concentration dependent. We explain this effect by the dynamics of the HIV-1 PR flexible flaps that cover the peptide substrate binding site and are crucial for enzyme activity, and by a possibly slower substrate–enzyme association time in the crowded conditions. Electronic supplementary material The online version of this article (10.1007/s00249-019-01392-1) contains supplementary material, which is available to authorized users.

Inter-related mechanical, thermal, and optical macroscopic properties of biomaterials are defined at the nanoscale by their constituent structures and patterns, which underpin complex functions of an entire bio-object. Here, the temperature diffusivity of a cicada (Cyclochila australasiae) wing with nanotextured surfaces was measured using two complementary techniques: a direct contact method and IR imaging. The 4-6-μm-thick wing section was shown to have a thermal diffusivity of α⊥ = (0.71 ± 0.15) × 10(-7) m(2)/s, as measured by the contact temperature wave method along the thickness of the wing; it corresponds to the inherent thermal property of the cuticle. The in-plane thermal diffusivity value of the wing was determined by IR imaging and was considerably larger at α∥ = (3.6 ± 0.2) × 10(-7) m(2)/s as a result of heat transport via air. Optical properties of wings covered with nanospikes were numerically simulated using an accurate 3D model of the wing pattern and showed that light is concentrated between spikes where intensity is enhanced by up to 3- to 4-fold. The closely packed pattern of nanospikes reduces the reflectivity of the wing throughout the visible light spectrum and over a wide range of incident angles, hence acting as an antireflection coating.

Thermal diffusivity of silk fibroin films, α = (1.6 ± 0.24) × 10−7 m2 s−1, was measured by a direct contact method. It was shown to be reduced down to ∼1 × 10−7 m2 s−1 in the crystallized phase, consistent with the multi-domain composition of β-sheet assemblies. Crystalline silk with β-sheets was made by dipping into alcohol and was used as a positive electron beam lithography (EBL) resist. It is shown by direct IR imaging of the 1619 cm−1 amide-I CO spectral signature and 3290 cm−1 amide-A N–H stretching band that an e-beam is responsible for unzipping β-sheets, which subsequently results in exposed areas returning to a water soluble state. This makes it possible to develop a water-based biocompatible silk resist and use it in lithography applications. The general principles of protein crystallization, traceable to spectral changes in IR amide bands of silk, can be used as a guide for the creation of new protein EBL resists and to quantify the electron dose required for solubility. Foam formation and laser treatments of silk can provide new approaches in surface functionalization and fabrication of 3D bio-scaffolds.

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis-Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37°C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis-Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.

Proteinase 3 (PR3) is a neutrophil serine protease present in cytoplasmic granules but also expressed at the neutrophil surface where it mediates proinflammatory effects. Studies of the underlying molecular mechanisms have been hampered by the lack of inhibitors of the PR3 membrane anchorage. Indeed while there exist inhibitors of the catalytic activity of PR3, its membrane interfacial binding site (IBS) is distinct from its catalytic site. The IBS has been characterized both by mutagenesis experiments and molecular modeling. Through docking and molecular dynamics simulations we have designed d-peptides targeting the PR3 IBS. We used surface plasmon resonance to evaluate their effect on the binding of PR3 to phospholipid bilayers. Next, we verified their ability of binding to PR3 via fluorescence spectroscopy and isothermal titration calorimetry. The designed peptides did not affect the catalytic activity of PR3. A few peptides bound to PR3 hydrophobic pockets and inhibited PR3 binding to lipids. While the (KFF)3K d-peptide inconveniently showed a significant affinity for the lipids, another d-peptide (SAKEAFFKLLAS) did not and it inhibited the PR3-membrane binding site with IC50 of about 40μM. Our work puts forward d-peptides as promising inhibitors of peripheral protein-membrane interactions, which remain high-hanging fruits in drug design.

International audience; Nanoparticles synthesized by laser ablation of bulk target materials in liquids have ligand-free surfaces since no chemical precursors are used for their synthesis, and thus, they are ideally suited for applications in the fabrication of organic solar cells in which the properties of the interface between the nanoparticles and the polymer blend matrix largerly determine the exciton splitting and transport of carriers to the external electrodes, properties crucial for the device operation and performance. Narrow band gap semiconducting quantum dots can act as sensitizers, increasing the absorption of the device active layer in the infrared part of the solar spectrum. In this work, a bulk PbS target was laser ablated (450 fs, 1,064 nm, 1 kHz) in ethanol for the synthesis of nanoparticle colloidal solutions. The solutions exhibit a broad absorption which extends at the longest wavelength measured of similar to 1,700 nm and beyond. The nanoparticles were directly mixed with the blend P3HT:PCBM for the formation of nanocomposites. The nanocomposites with the nanoparticles exhibit lower transmission in the whole spectral range as compared to the blend without the nanoparticles.

A series of chiral A4, A2B2, and AB3 porphyrins bearing proline moieties at the meso-phenyl group has been synthesized. Photostability studies revealed that the number of L-proline units and their position on meso-phenyl rings strongly influence the decomposition rate of the catalyst. 5,10,15-Tris(mesityl)-20-(3-prolinanilidylphenyl)-21,23H-porphyrin is the most stable while porphyrin bearing four 3-prolinanilidylphenyl substituents completely decomposes in CHCl 3 within 3 h. Singlet oxygen quantum yields of the conjugates were determined by measuring the peak areas of the NIR emission of 1 O 2 (1280 nm) generated by these compounds and compared to that generated by the reference standard TPP. Selected porphyrins were tested as catalysts in the photooxidation of carbonyl compounds at the α-position.

An effective and reliable method was developed for the synthesis of protoporphyrin IX-derived amides. The reaction of protoporphyrin IX with an amino acid [ l -leucine, l -valine, l -lysine, l -aspartic acid (Asp), l -glutamic acid (Glu), glycine, or 6-aminohexanoic acid] or a short peptide (Asp-Glu) as an amine counterpart in the presence of O -(benzotriazol-1-yl)- N , N , N ′, N ′-tetramethyluronium hexafluorophosphate and 1 H -1,2,3-benzotriazol-1-ol gave the desired diamides in excellent yields.

Fluorescence polarisation immunoassays (FPIAs) based on monoclonal antibodies for detection of three strobilurin fungicides - kresoxim-methyl (KM), trifloxystrobin (TF) and picoxystrobin (PC), were developed and optimised. Fluorescein-labelled derivatives of target antigens (tracers) were synthesised and purified by thin-layer chromatography. Influence of tracer structures on the assay parameters was investigated. For KM and TF, the best assay performance was achieved with the homologous pairs of reagents. For the PC assay, the heterologous tracer, i.e. fluorescein-labelled derivative of TF, was used. The developed FPIAs were applied to the determination of KM, TF and PC in red wine. Most optimal sample preparation was achieved with cross-linked poly(vinylpyrrolidone) as a sorbent. This clean-up is simple, rapid and allows determination of all three strobilurin fungicides in one sample. Detection limits of the developed FPIAs in red wine were 28, 6 and 5 ng/mL for KM, TF and PC, respectively. Recovery in spiked samples averaged between 80% and 104%. Intra- and inter-assay coefficients of variance were less than 12%. The developed FPIA methods can be applied to screening of wine samples for KM, TF and PC residues without complicated cleanup.

Silk patterns in a film of amorphous water-soluble fibroin are created by tailored exposure to femtosecond-laser pulses (1030 nm/230 fs) without the use of photo-initiators. This shows that amorphous silk can be used as a negative tone photo-resist. It is also shown that water insoluble crystalline silk films can be precisely ablated from a glass substrate achieving the patterns of crystalline silk gratings on a glass substrate. Bio-compatible/degradable silk can be laser structured to achieve conformational transformations as demonstrated by infrared spectroscopy.

Convenient synthesis of new methacryloyloxy-substituted phthalocyanines for polymerization reactions, i.e. copper(II) 2,9,16,23-tetra(methacryloxyethoxycarbonyl)phthalocyanine and copper 2,9,16-tris(tert-butyl)-23-(methacryloxyethoxycarbonyl)phthalocyanine, consisting of just a few steps, has been developed. The copolymerization with a dimethacrylate derivative was investigated. The substituted phthalocyanines are interesting materials for ophthalmosurgery in ophthalmology (contact vision correction of eye lenses).

We report a drastic enhancement of electrocatalytic activity toward glucose oxidation by using novel electrocatalysts on the basis of “bare” unprotected Au nanoparticles synthesized by methods of laser ablation in pure deionized water. The recorded current density of 2.65 A cm–2 mg–1 for glucose electrooxidation was higher than a relevant value for conventional chemically synthesized Au nanoparticles by an order of magnitude and outperformed all data reported in the literature for metal and metal alloy-based electrocatalysts. The enhanced electrocatalytic characteristics of laser-synthesized nanoparticles are explained by the absence of any organic contaminants or protective ligands on their surface, the relatively small size of nanoparticles, and their particular crystallographic structure. The employment of bare nanomaterials in glucose electrooxidation schemes promises a radical improvement in current biofuel cell technology and its successful application in bioimplantable devices.

International audience; We report a size-controllable synthesis of stable aqueous solutions of ultrapure low-size-dispersed Au nanoparticles by methods of femtosecond laser fragmentation from preliminary formed colloids. Such approach makes possible the tuning of mean nanoparticle size between a few nm and several tens of nm under the size dispersion lower than 70% by varying the fluence of pumping radiation during the fragmentation procedure. The efficient size control is explained by 3D geometry of laser fragmentation by femtosecond laser-induced white light super-continuum and plasma-related phenomena. Despite the absence of any protective ligands, the nanoparticle solutions demonstrate exceptional stability due to electric repulsion effect associated with strong negative charging of formed nanoparticles. Stable aqueous solutions of bare gold nanoparticles present a unique object with a variety of potential applications in catalysis, surface-enhanced Raman spectroscopy, photovoltaics, biosensing and biomedicine.

5th European-Materials-Research-Society Symposium on Laser Material Interactions for Micro- and Nano- Applications, Strasbourg, FRANCE, MAY 27-31, 2013; International audience; The experimental conditions were investigated enabling one to fabricate Au nanowire networks by pulsed laser ablation in water. The study revealed that it is possible to produce alternatively nanoparticles (or aggregates) or nanowire networks at certain wavelengths depending on the laser fluence. An Au disc immersed in double-distilled water was used as a target. The second (lambda(SHG) = 532 nm) and the third lambda(THG) = 355 nm) harmonics of a Nd:YAG laser system were utilized to produce different Au colloids. The values of the laser fluence for both wavelengths under the experimental conditions chosen were varied from several J/cm(2) to tens of J/cm(2). The optical extinction spectra of the colloids in the UV/vis region were obtained to evaluate the structure of the dispersed Au phase. Transmission electron microscopy (TEM) was applied to visualize the size and morphology of the colloidal particles. Their structure and phase composition were studied by high-resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) and used to make an assumption on how they had been formed. (C) 2014 Elsevier B.V. All rights reserved.

Reda Kubiliūtė,1,2 Ksenia A Maximova,3 Alireza Lajevardipour,1 Jiawey Yong,1 Jennifer S Hartley,1 Abu SM Mohsin,1 Pierre Blandin,3 James WM Chon,1 Marc Sentis,3 Paul R Stoddart,1 Andrei Kabashin,3 Ričardas Rotomskis,2 Andrew HA Clayton,1,4 Saulius Juodkazis1,4 1Centre for Micro-Photonics and Industrial Research Institute Swinburne, Faculty of Engineering and Industrial Sciences Swinburne University of Technology, Hawthorn, VIC, Australia; 2Laboratory of Biomedical Physics, Vilnius University Institute of Oncology, Baublio, Vilnius, Lithuania; 3Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Lasers, Plasmas and Photonics Processing Laboratory, Campus de Luminy, Marseille, France; 4The Australian National Fabrication Facility, Victoria node, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, VIC, Australia Abstract: Aqueous solutions of ultra-pure gold nanoparticles have been prepared by methods of femtosecond laser ablation from a solid target and fragmentation from already formed colloids. Despite the absence of protecting ligands, the solutions could be (1) fairly stable and poly size-dispersed; or (2) very stable and monodispersed, for the two fabrication modalities, respectively. Fluorescence quenching behavior and its intricacies were revealed by fluorescence lifetime imaging microscopy in rhodamine 6G water solution. We show that surface-enhanced Raman scattering of rhodamine 6G on gold nanoparticles can be detected with high fidelity down to micromolar concentrations using the nanoparticles. Application potential of pure gold nanoparticles with polydispersed and nearly monodispersed size distributions are discussed. Keywords: nanotechnologies applications, methods of nanofabrication and processing, materials for nanomedicine

International audience; An ultrashort laser-assisted method for fast production of concentrated aqueous solutions of ultrapure Si-based colloidal nanoparticles is reported. The method profits from the 3D geometry of femtosecond laser ablation of water-dispersed microscale colloids, prepared preliminarily by the mechanical milling of a Si wafer, in order to avoid strong concentration gradients in the ablated material and provide similar conditions of nanocluster growth within a relatively large laser caustics volume. We demonstrate the possibility for the fast synthesis of non-aggregated, low-size-dispersed, crystalline Si-based nanoparticles, whose size and surface oxidation can be controlled by changing the initial microcolloid concentration and the amount of dissolved oxygen in the water. Due to their much superior purity compared to the chemically synthesized counterparts and their photoluminescence response, the nanoparticles present the possibility for biological in vivo applications such as drug vectoring, imaging, and therapeutics.

Laser-printing of crystalline silk patterns in amorphous fibroin film is demonstrated by femtosecond-laser pulses 1030 nm/230 fs without use of photo-initiator. The same patterns can be achieved by ablation of the crystalline silk film. Amorphous silk can be used as a negative tone photo-resist.