Synthesis and study of new siderophore analog-ciprofloxacin conjugates with antibiotic activities against Pseudomonas aeruginosa and Burkholderia spp.

Antibacterial resistance is a healthcare burden. Among Gram-negative bacteria, Pseudomonas aeruginosa belongs to the first list of antibiotic-resistant "priority pathogens" described by the World Health Organization. Formerly Pseudomonas pseudomallei, Burkholderia pseudomallei, responsible for melioidosis, is considered as a potential bioterrorist weapon by the Centers of Diseases Control and Prevention. We are interested in the development of new ways to combat these bacteria, targeted due to their high level of resistance to antibiotics via a lack of membrane permeability or efflux. Using iron transport systems is a promising strategy to bypass the bacteria cell membrane and restore the activity of conventional antibiotics such as ciprofloxacin. Specific outer membrane receptors are necessary to most microbes as they allow iron uptake, essential for their survival through siderophore-dependent mechanisms. These systems may allow the introduction of antibacterial agents, chemically coupled to a natural or synthetic siderophore molecule to form siderophore-antibiotic conjugates. In this work, we describe the synthesis of six new siderophore analog-ciprofloxacin conjugates including cleavable linker or not. The siderophore analogs correspond to a mono-catechol or a hydroxypyridinone moiety recognized by both Pseudomonas and Burkholderia species. Physico-chemical studies showed that (i) conjugates were unable to interact or cross the membrane by passive diffusion and (ii) conjugates with cleavable linker are stable in physiologic environment. Biological evaluations have highlighted a promising compound 2d, bearing an hydroxypyridinone moiety with a cleavable linker, active on a large panel of strains of Pseudomonas aeruginosa, Burkholderia pseudomallei and Burkholderia thailandensis without toxicity observed in vitro.

Biomimicry of the flexor digitorum superficialis: Systematic literature review.

Biomimicry consists in imitating nature to solve complex human problems. The hand surgeon usually tries to copy and recreate the structure-to-function and function-to-control relationships of the native tissues after damage. With its exceptional structure and biomechanics, the flexor digitorum superficialis (FDS) has been an important source of inspiration for artificial hand system reconstruction. The present systematic literature review highlights the twenty-two artificial hand system reconstructions derived from the FDS, and presents biomimicry as an alternative approach in clinical research in hand surgery.

Enzyme-assisted mineralization of calcium phosphate: exploring confinement for the design of highly crystalline nano-objects.

In hard tissues of vertebrates, calcium phosphate (CaP) biomineralization is a fascinating process that combines specific physicochemical and biochemical reactions, resulting in the formation of extracellular matrices with elegant nanoarchitectures. Although several "biomimetic" strategies have been developed for the design of mineralized nanostructured biointerfaces, the control of the crystallization process remains complex. Herein, we report an innovative approach to overcome this challenge by generating, in situ, CaP precursors in a confined medium. For this purpose, we explore a combination of (i) the layer-by-layer assembly, (ii) the template-based method and (iii) the heterogeneous enzymatic catalysis. We show the possibility of embedding active alkaline phosphatase in a nanostructured multilayered film and inducing the nucleation and growth of CaP compounds under different conditions. Importantly, we demonstrate that the modulation of the crystal phase from spheroid-shaped amorphous CaP to crystalline platelet-shaped hydroxyapatite depends on the degree of confinement of active enzymes. This leads to the synthesis of highly anisotropic mineralized nanostructures that are mechanically stable and with controlled dimensions, composition and crystal phase. The present study provides a straightforward, yet powerful, way to design anisotropic nanostructured materials, including a self-supported framework, which may be used in broad biomedical applications.

Calcium phosphate mineralization through homogenous enzymatic catalysis: Investigation of the early stages.

HYPOTHESIS: The crystallization of calcium phosphate (CaP) is a ubiquitous process that occurs in several mineralized tissues and involves a variety of biochemical and chemical reactions. This issue has been hitherto continuously studied from supersaturated solutions (chemical procedure), i.e. by adding calcium and orthophosphate ions in a homogenous phase. Yet, both in vivo and in vitro investigations have clearly shown the implication of enzymes, namely alkaline phosphatase (ALP), to initiate the mineralization process by generating orthophosphate ions. EXPERIMENTS: We report a thorough investigation on the mechanism of enzyme-induced mineralization in homogenous phase (enzymatic procedure). For this purpose, ALP is introduced in Ca2+/Mg2+-containing solution (pH = 7.4; 37 °C), and its activity modulated by the concentration of its substrate. FINDINGS: Results show that after 24 h of mineralization both chemical and enzymatic procedures lead to the formation of well-crystalline hydroxyapatite nano-objects, however with noticeable impact on their shape and dimensions. Remarkably enough, by combining in situ monitoring and ex situ characterizations, we identify several intermediate phases, including amorphous phase, dicalcium phosphate dehydrate phase (DCPD or brushite) and Whitlockite (WH). Besides, mineralized nano-objects with a core-shell structure is observed, and hydroxyapatite platelets are shown to grow on the surface of their shell.

Enzymatic approach in microbial-influenced corrosion: a review based on stainless steels in natural waters.

The electrochemical behavior of stainless steels (SS) in natural waters is characterized by the ennoblement of their free corrosion potential (E(corr)). This phenomenon depends strongly on the settlement of biofilms on SS surfaces. Many hypotheses have been proposed to explain the biofilm action, in particular the enzymatic catalysis plays an important role by shifting the cathodic and/or anodic processes. However, there are still only few studies relating the use of purified enzymes. In contrast with bacteria-associated corrosion, the direct influence of enzymes is still poorly documented. The aim of this review is to show the benefits of the enzymatic approach in the study of biocorrosion. Indeed, enzymatic systems may constitute convenient models to mimic microbial influenced corrosion and to evaluate the behavior of metallic materials in natural waters.

Ennoblement of stainless steel in the presence of glucose oxidase: nature and role of interfacial processes.

The ennoblement of the free corrosion potential (E(corr)) of AISI 316L stainless steel which did not occur in synthetic fresh water (SFW), was observed after introduction of glucose oxidase (Gox) and glucose, or of hydrogen peroxide (H(2)O(2)). The composition of the surface was monitored using AFM and XPS, a detailed XPS analysis being based on the discrimination between oxygen of organic and inorganic nature proposed in a previous study. In H(2)O(2) medium, the main changes regarding the inorganic phase were the increase of the oxygen concentration in the passive film, the increase of the molar concentration ratio of oxidized species Fe(ox)/Cr(ox) and the growth of nanoparticles, presumably made of ferric oxide/hydroxide. In Gox medium, no significant changes were observed in both oxygen concentration and Fe(ox)/Cr(ox) ratio, but the density of colloidal particles decreased, indicating a dissolution of Fe oxide/hydroxide under the influence of gluconate. In contrast with H(2)O(2), in SFW and Gox the amount of organic compounds increased due to the accumulation of polysaccharides and proteins. The influence of glucose oxidase on the ennoblement of stainless steel is not due to indirect effects of H(2)O(2) through the change of surface composition. The E(corr) ennoblement seems to be directly due to the presence of H(2)O(2) and to the electrochemical behavior of H(2)O(2) and related oxygen species. This consideration is important for understanding and controlling microbial influenced corrosion.

Evolution of the passive film and organic constituents at the surface of stainless steel immersed in fresh water.

The evolution of the surface of a conventional stainless steel (AISI 316L) immersed in aqueous medium simulating fresh water (pH approximately 8) was studied using XPS and AFM. A detailed analysis of XPS spectra allowed a distinction to be made between oxygen of organic and inorganic nature. During the first 48 h, the main changes concern the inorganic phase: the oxygen concentration in the passive layer increases, owing both to oxidation of metal elements, including conversion of Fe(II) to Fe(III), and to hydration; the molar ratio of oxidized species Fe(ox)/Cr(ox) decreases slightly; the formation of colloidal particles, presumably made of ferric hydroxide, is observed by AFM. After longer periods of immersion, the Fe(ox)/Cr(ox) is higher, while the coating of colloidal particles reaches a full surface coverage. The amount of organic compounds increases further and the XPS spectra reveal the accumulation of polysaccharides and proteins, which indicate that these organic compounds are of biological origin.

Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1.

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.

Sample preparation procedures for biological atomic force microscopy.

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three-dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.

The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of P-glycoprotein.

A new series of potential flavonoidic modulators of P-glycoprotein activity has been prepared. The flavanolignan silybin was first oxidised to dehydrosilybin and then C-alkylated with either prenyl or geranyl bromide. The resulting isoprenoid dehydrosilybins were shown to display high in vitro affinities for direct binding to P-glycoprotein, which ranged them among the best flavonoids ever tested.