Aline Percot - Independent Researcher (original) (raw)

Thesis Chapters by Aline Percot

Fibrous proteins (keratin, elastin, collagen, fibroin ...) make up to one third of the proteins o... more Fibrous proteins (keratin, elastin, collagen, fibroin ...) make up to one third of the proteins of mammals and birds. They are structural proteins with a protective and/or mechanical function. Silk appears to be the ‘simplest’ model because it mainly consists of two small amino acids residues (alanine and glycine). Some silks have comparable or superior mechanical properties compared to those of synthetic fibres and could be used in technical applications (e.g. biomedical) if the variability of their properties can be controlled. This work focuses on the structure of silks from: Bombyx mori (domestic silkworm) degummed or not, Nephila madagascariensis (wild spider, no sericin coating), GM Bombyx mori (including a gene of Nephila) a recombinant spider silk 4RepCT (Escherichia Coli). Silk is analyzed by Raman spectroscopy (and FTIR), uni-axial tensile testing, and also by the coupling of these methods. The analysis of the low wavenumbers region in Raman spectroscopy allowed the characterization of ordered regions of 2-3 microns separated by about 60 microns. This is the first evidence of the heterogeneous structure of silk. Coupling with the uni-axial tensile test shows that these ordered regions are stressed under macroscopic deformation, suggesting silk organization according to Prevorsek’s model, i.e. that the same macromolecular chain belongs to both amorphous and ordered regions. The statistical study of the mechanical properties of silkworm and spider silks shows great dispersion, but a good stability over time (decades). Genetic modification does not improve the fibres mechanical properties but a slight decrease in their variability. Various strategies have been investigated to control the variability: bacterial production, solubilization of silk and films regeneration. The role of water in silk biosynthesis, as well as the effect of various parameters (filtration, pH, drying ...) during the preparation of the films were studied. It was confirmed that the presence of protein aggregates promotes the organization in film and two types of films were prepared. The most amorphous ones have the most interesting mechanical properties, though only a few percent of those from the starting fibres. The fabrication of regenerated silk matrix composites reinforced by silk fibres increases the strength and strain to failure. These initial results are encouraging for the development of silk fibres/regenerated silk matrix composite materials.

Papers by Aline Percot

Contactless approach through portable vibrational spectroscopy to identify the binders of German and Italian Modern Paintings

Order variations along silkworm/spider silk

La soie, 'modèle' de polymères naturels fibreux : analyse vibrationnelle et nano/micromécanique, de la fibre au composite - Silk, 'pattern' of natural fibrous polymer: vibrationnal and nano/micromechanical analysis from fibre to composite

Talanta, 2021

Highlights:-Non-invasive mobile approach to characterise artist materials implemented in Modern p... more Highlights:-Non-invasive mobile approach to characterise artist materials implemented in Modern paintings-Validation of Specular Reflectance Infrared Spectroscopy analyses to identify paint binders-Combination of Raman and infrared spectroscopy for identifying Modern paints binders and pigments-Contactless spectroscopic identification of the binders and pigments of polymeric paints-On-site analytical study of artworks by German and Italian painters of the 1960s Abstract. Seven artworks representing the diversity of paints used around the 1960s and created by German and Italian painters (J. Albers, A. Bonalumi, L. Boille, T. Scialoja and M. Schifano) were studied onsite at the Galleria Nazionale d'Arte Moderna (Rome) with mobile instruments. We present a methodology based on Specular Reflectance Infrared Spectroscopy (SR-FTIR) adapted to unvarnished paintings. Complementary measurements have been performed by Raman spectroscopy. Characteristic bands regarding as-recorded infrared reflectance spectra and Kramers-Kronig Transformationconverted absorbance spectra are identified according to literature and reference spectra recorded on representative commercially available paints. To distinguish the different binders by SR-FTIR, we propose spectroscopic markers as the comparison of the intensity of carbonyl band around 1730-1735 cm-1 with bands at ~1160 (for acrylic), ~1230 (for PVAc), and 1270 cm-1 (for alkyds). On the other hand, oil/resin binders are characterized by intense and thin CH 2 , CH 3 IR absorption bands around 2920-2850 cm-1 , combined with an intense 1260 cm-1 band and a characteristic concave cradle shape (between ca. 1750 and 1260 cm-1). The results obtained establish the relevance of the implemented mobile non-invasive infrared spectroscopy analytical approach by successfully identifying acrylic, vinylic, oil media and enamel paints, with or without opacifiers, which is supplemented by Raman analyses for pigment identification.

International Journal of Biological Macromolecules, 2020

Fibrin-based gels are used in clinics as biological glues but their application as 3D cellularize... more Fibrin-based gels are used in clinics as biological glues but their application as 3D cellularized scaffolds is hindered by processing and stability issues. Silicification of fibrin networks appears as a promising strategy not only to address these limitations but also to take advantage of the bioactivity of Si. However, it raises the question of the influence of silica sources on fibrin self-assembly. Here tetraethoxysilane, aminopropyltriethoxysilane and silica nanoparticles were used to design hybrid and nanocomposite fibrin-based hydrogels. By varying the concentration in silica source, we could evidence two regimes of interactions that depend on the extent of inorganic condensation. These interactions modulated the fibrillar structure of the fibrin network from more than 500 nm to less than 100 nm. These nanofibrillar hydrogels could exhibit higher mechanical properties than pure fibrin while preserving their capacity to support proliferation of myoblasts, opening promising perspectives for the use of fibrin-silica constructs in tissue engineering.

Zoomorphology, 2020

Development and evolution of the shell in cephalopods is difficult to establish as there is few s... more Development and evolution of the shell in cephalopods is difficult to establish as there is few species with a calcified shell that could be fossilized (stable in geological time). Internal cuttlebone of sepiids is so particular that homologies are difficult to find. The developmental sequence in embryos give some response elements by comparison with adult cuttlebone. The macro and microstructure of adult shell is well known but an approach at nanostructural level allows to determine structure and composition of the two main parts, the dorsal shield and chambered part. We evidence in the embryonic shell, mainly organic, a light calcification of the shell, which occurs directly as aragonite, as it is all along the formation of the shell and whatever the parts. In embryonic shell, the prismatic and/or lamellar layers, present in adult, are not differentiated and the dorsal shield grows progressively, from posterior to anterior. Despite microstructural differences, all layers of both chambered part and dorsal shield are composed of rounded nanogranules (between 50 and 100 nm), similar to what is found in other mollusc shells. Finally, the presence of pillars evidenced in embryo suggests either that their absence in extinct Manuscript Click here to access/download;Manuscript;YD-LBP-GL-Sepia-R1.docx Click here to view linked References 2 lineages of sepiids is the result of a diagenetic process or that they are a novelty in present sepiid species.

European Journal of Organic Chemistry, 2018

We took advantage of the powerful adenine SERS (Surface Enhanced Raman Spectroscopy) probe to des... more We took advantage of the powerful adenine SERS (Surface Enhanced Raman Spectroscopy) probe to design peptideadenine conjugates as candidates for their use as serine protease substrates. Whereas the direct introduction of the peptide sequence on the adenine exocyclic N6 amine gave an imidazopurinone derivative, the introduction of an aminoethyl linker between the adenine group and the peptide chain led to the expected candidate probes. These potential substrates were then evaluated for the monitoring of the hydrolytic activity of trypsin, used as a model of protease, by HPLC and by SERS. We demonstrated that Boc-VPRadenine conjugate was a substrate of trypsin and constitute a good starting point to design optimized substrates to monitor the protease activity by SERS.

Journal of Composite Materials, 2017

Silk is a biocompatible material with remarkable mechanical properties. A variety of composite ma... more Silk is a biocompatible material with remarkable mechanical properties. A variety of composite materials using silk as a matrix or for reinforcement have been prepared for many biological applications. However, although a range of composites containing silk have been designed, the mechanical properties achieved for the regenerated silk (solubilisation + film/matrix or fibre production) remain relatively weak (strength: 30–50 MPa). In this study, by combining the high ultimate strength (up to 1 GPa) of natural silk fibres and the chemical properties of regenerated silk, we developed a composite based on silk alone. We investigated how to improve the tensile mechanical properties of silk matrices derived from Bombyx mori silkworms by film processing and by incorporating silk fibres, either natural or degummed, that is, without the sericin coating. The structure and orientation modifications of fibroin macromolecules within the matrix were monitored by Raman spectroscopy; unexpectedly ...

Journal of Raman Spectroscopy, 2016

Proteases are found deregulated in many diseases including cancer and neurodegenerative diseases.... more Proteases are found deregulated in many diseases including cancer and neurodegenerative diseases. They thus represent good therapeutical targets for the development of inhibitors mainly small organic molecules. Peptide substrates containing fluorogenic groups constitute central tools for the monitoring of protease activities and inhibitor screening platforms. Amino-methyl coumarin (AMC) is a well-known fluorogenic group that functionalized a huge number of peptide substrates used for kinetics in vitro but also in vivo. However, either autofluorescence or quenching of the AMC fluorescence could compromise selection and accurate evaluation of these inhibitors. It is thus needed to explore alternative spectroscopic tools to unravel these limitations. Here, we investigate whether AMC could constitute a valuable Surface Enhanced Raman Spectroscopy probe in the presence of Creighton' silver colloids under 532-nm excitation to monitor protease activity and to evaluate inhibitors. The kallikrein-related peptidase 8 was used as model of proteolytic enzyme. Band-Target Entropy Minimization analysis was successfully used to validate the present Surface Enhanced Raman Spectroscopy approach.

Journal of structural biology, Jan 6, 2016

During premolt, crayfish develop deposits of calcium ions, called gastroliths, in their stomach w... more During premolt, crayfish develop deposits of calcium ions, called gastroliths, in their stomach wall. The stored calcium is used for the calcification of parts of the skeleton regularly renewed for allowing growth. Structural and molecular analyses of gastroliths have been primarily performed on three crayfish species, Orconectes virilis, Procambarus clarkii, and more recently, Cherax quadricarinatus. We have performed high-resolution analyses of gastroliths from the native noble crayfish, Astacus astacus, focusing on the microstructure, the mineralogical and elemental composition and distribution in a comparative perspective. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) observations showed a classical layered microstructure composed of 200-nm diameter granules aligned along fibers. These granules are themselves composed of agglomerated nanogranules of 50nm-mean diameters. Denser regions of bigger fused granules are also present. Micro-Raman ...

Journal of Proteomics, 2017

Protein compounds constituting mollusk shells are known for their major roles in the biomineraliz... more Protein compounds constituting mollusk shells are known for their major roles in the biomineralization processes. These last years, a great diversity of shell proteins have been described in bivalves and gastropods allowing a better understanding of the calcification control by organic compounds and given promising applications in biotechnology. Here, we analyzed for the first time the organic matrix of the aragonitic Sepia officinalis shell, with an emphasis on protein composition of two different structures: the dorsal shield and the chambered part. Our results highlight an organic matrix mainly composed of polysaccharide, glycoprotein and protein compounds as previously described in other mollusk shells, with quantitative and qualitative differences between the dorsal shield and the chamber part. Proteomic analysis resulted in identification of only a few protein compounds underlining the lack of reference databases for Sepiidae. However, most of them contain domains previously characterized in matrix proteins of aragonitic shell-builder mollusks, suggesting ancient and conserved mechanisms of the aragonite biomineralization processes within mollusks. Significance The cuttlefish's inner shell, better known under the name "cuttlebone", is a complex mineral structure unique in mollusks and involved in tissue support and buoyancy regulation. Although it combines useful properties as high compressive strength, high porosity and high permeability, knowledge about organic compounds involved in its building remains limited. Moreover, several cuttlebone organic matrix studies reported data very different from each other or from other mollusk shells. Thus, this study provides 1) an overview of the organization of the main mineral structures found in the S. officinalis shell, 2) a reliable baseline about its organic composition, and 3) a first descriptive proteomic approach of organic matrices found in the two main parts of this shell. These data will contribute to the general knowledge about mollusk biomineralization as well as in the identification of protein compounds involved in the Sepiidae shell calcification.

Journal of Polymer Science Part B: Polymer Physics, 2000

Linear and crosslinked polymers based on N-isopropylacrylamide (NIPAAm) exhibit unusual thermal p... more Linear and crosslinked polymers based on N-isopropylacrylamide (NIPAAm) exhibit unusual thermal properties. Aqueous solutions of poly(N-isopropylacrylamide) (PNIPAAm) phase-separate upon heating above a lower critical solution temperature (LCST), whereas related hydrogels undergo a swelling-shrinking transition at an LCST. A linear copolymer made of NIPAAm/acryloxysuccinimide (98/2 mol/mol) and two hydrogels with different hydrophilicities were prepared. Fourier transform infrared (FTIR) spectroscopy was employed to determine the transition temperature and provide insights into the molecular details of the transition via probing of characteristic bands as a function of temperature. The FTIR spectroscopy method described here allowed the determination of the transition temperature for both the linear and crosslinked polymers. The transition temperatures for PNIPAAm and the gel resulting from the crosslinking with polylysine or N,NЈ-methylenebisacrylamide (MBA) were in the same range, 30-35°C. For the gels, the transition temperature increased with the hydrophilicity of the polymer matrix. The spectral changes observed at the LCST were similar for the free chains and the hydrogels, implying a similar molecular reorganization during the transition. The COH stretching region suggests that the N-isopropyl groups and the backbone both underwent conformational changes and became more ordered upon heating above the LCST. An analysis of the amide I band suggests that the amide groups of the linear polymer were mainly involved in hydrogen bonding with water molecules below the LCST, the chain being flexible and disordered in a water solution. During the transition, around 20% of these intermolecular hydrogen bonds between the polymer and water were broken and replaced by intramolecular hydrogen bonds. Similar changes were also observed at the LCST of a gel crosslinked with MBA.

Design and characterization of anchoring amphiphilic peptides and their interactions with lipid vesicles

Biopolymers, 1999

In an effort to develop a polymer/peptide assembly for the immobilization of lipid vesicles, we h... more In an effort to develop a polymer/peptide assembly for the immobilization of lipid vesicles, we have made and characterized four water-soluble amphiphilic peptides designed to associate spontaneously and strongly with lipid vesicles without causing significant leakage from anchored vesicles. These peptides have a primary amphiphilic structure with the following sequences: AAAAAAAAAAAAWKKKKKK, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK, and KKAALLLAAAAAAAAAAAAAAAAAAAWKKKKKK and its reversed homologue KKKKKKWAAAAA AAAAAAAAAAAAAALLLAAKK. Two of the four peptides have their hydrophobic segments capped at both termini with basic residues to stabilize the transmembrane orientation and to increase the affinity for negatively charged vesicles. We have studied the secondary structure and the membrane affinity of the peptides as well as the effect of the different peptides on the membrane permeability. The influence of the hydrophobic length and the role of lysine residues were clearly established. First, a hydrophobic segment of 24 amino acids, corresponding approximately to the thickness of a lipid bilayer, improves considerably the affinity to zwitterionic lipids compared to the shorter one of 12 amino acids. The shorter peptide has a low membrane affinity since it may not be long enough to adopt a stable conformation. Second, the presence of lysine residues is essential since the binding is dominated by electrostatic interactions, as illustrated by the enhanced binding with anionic lipids. The charges at both ends, however, prevent the peptide from inserting spontaneously in the bilayer since it would involve the translocation of a charged end through the apolar core of the bilayer. The direction of the amino acid sequence of the peptide has no significant influence on its behavior. None of these peptides perturbs membrane permeability even at an incubation lipid to peptide molar ratio of 0.5. Among the four peptides, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK is identified as the most suitable anchor for the immobilization of lipid vesicles.

Biophysical Journal, 2001

Several studies on intact and model stratum corneum (SC), the top layer of the epidermis, have su... more Several studies on intact and model stratum corneum (SC), the top layer of the epidermis, have suggested the presence of crystalline domains. In the present work, we used micro-Raman mapping to detect lipid domains in model lipid mixtures formed by an equimolar mixture of ceramides, cholesterol, and palmitic acid, the three main lipid species of SC. We were able to determine the spatial distribution of the three compounds individually based on the systematic analysis of band areas. As a control, we studied freeze-dried lipid mixtures, and the Raman microspectroscopy reported faithfully the homogeneous distribution of the three compounds. Spectral mapping was then performed on hydrated equimolar mixtures carefully annealed. In this case, clear phase separations were observed. Domains enriched in cholesterol, ceramides, or palmitic acid with a size of a few tens of square microns were detected. These findings constitute the first direct evidence of the formation of heterogeneous domains in the SC lipid models in a bulk phase. Raman microspectroscopy is an innovative approach to characterize the conditions leading to the formation of domains and provides new insights into the understanding of the skin barrier.

Bioconjugate Chemistry, 2000

This amphiphilic peptide was previously designed in our group to interact spontaneously and stron... more This amphiphilic peptide was previously designed in our group to interact spontaneously and strongly with vesicles without perturbing their permeability. At the end of the solid-phase peptide synthesis, the peptide was left on the polymer beads and this novel polymer-peptide system was used for vesicle immobilization. It was shown that this polymer-peptide system could immobilize as much as 200 µmol of lipids per gram of dry resin. The amount of immobilized vesicles was decreased by a reduction of the proportion of the negatively charged lipids in the vesicles, indicating the importance of electrostatic interactions in the immobilization of the vesicles. The integrity of the vesicles was mostly preserved after the immobilization. This new polymerpeptide system was used easily and successfully to immobilize a membrane-bound enzyme, γ-glutamyl transpeptidase. The activity of the membrane-bound enzyme was studied by monitoring the release of p-nitroaniline. The activity of the enzyme was still retained, even after being re-used eight times, indicating the strong immobilization of the enzyme in its active form. The polymer-peptide support could be regenerated by washing with ethanol and reused.

Annals of the New York Academy of Sciences

In epithelia, claudin proteins are important components of the tight junctions as they determine ... more In epithelia, claudin proteins are important components of the tight junctions as they determine the permeability and specificity to ions of the paracellular pathway. Mutations in CLDN10 cause the rare autosomal recessive HELIX syndrome (Hypohidrosis, Electrolyte imbalance, Lacrimal gland dysfunction, Ichthyosis, and Xerostomia), in which patients display severe enamel wear. Here, we assess whether this enamel wear is caused by an innate fragility directly related to claudin-10 deficiency in addition to xerostomia. A third molar collected from a female HELIX patient was analyzed by a combination of microanatomical and physicochemical approaches This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Les 'meilleures' fibres de soie, polyamides naturels, présentent en dépit d'une très grande varia... more Les 'meilleures' fibres de soie, polyamides naturels, présentent en dépit d'une très grande variabilité une très bonne résistance à la rupture (200-1000 MPa, typiquement). La bonne biocompatibilité des films de fibroïne de Bombyx mori pour des applications biomédicales a été démontrée, la soie ayant autrefois été utilisée comme fil de suture. Le processus de dissolution dégrade les macromolécules et les propriétés mécaniques de la soie régénérée, contrainte à la rupture comme allongement, sont bien inférieures à celle des fibres initiales (< quelques % en contrainte/fibre). L'objectif est d'améliorer les propriétés mécaniques en traction de films de soie régénérée par l'incorporation de fibres, avec ou sans leur gaine de séricine. Des fibres décreusées (gaine de séricine éliminée) sont dissoutes et dialysées afin d'obtenir un liquide/gel plus ou moins visqueux qui par évaporation produit une matrice de soie. Les modifications de structure et d'orientation des macromolécules de fibroïne sont suivies par spectroscopie Raman. L'incorporation de quelques % (en volume) de fibres améliore la résistance et l'énergie dissipée à la rupture. Le comportement mécanique apparait très dépendant de la mise en oeuvre et de nombreux paramètres restent à optimiser.

Calcium Deposits in the Crayfish, Cherax quadricarinatus: Microstructure Versus Elemental Distribution

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada, Jan 28, 2016

The crayfish Cherax quadricarinatus stores calcium ions, easily mobilizable after molting, for ca... more The crayfish Cherax quadricarinatus stores calcium ions, easily mobilizable after molting, for calcifying parts of the new exoskeleton. They are chiefly stored as amorphous calcium carbonate (ACC) during each premolt in a pair of gastroliths synthesized in the stomach wall. How calcium carbonate is stabilized in the amorphous state in such a biocomposite remains speculative. The knowledge of the microstructure at the nanometer level obtained by field emission scanning electron microscopy and atomic force microscopy combined with scanning electron microscopy energy-dispersive X-ray spectroscopy, micro-Raman and X-ray absorption near edge structure spectroscopy gave relevant information on the elaboration of such an ACC-stabilized biomineral. We observed nanogranules distributed along chitin-protein fibers and the aggregation of granules in thin layers. AFM confirmed the nanolevel structure, showing granules probably surrounded by an organic layer and also revealing a second level of ...

Acta Pharmaceutica Sciencia

The alkaloid hordenine was firstly isolated from marine algae Phyllophora nervosa in 1969. In thi... more The alkaloid hordenine was firstly isolated from marine algae Phyllophora nervosa in 1969. In this work hordenine occurrence in Gelidium crinale collected from Sile is reported. The content of hordenine was 9.54 μg/g. This amt. is lower than in the other hordenine contg. algae. It is the first record of hordenine in Gelidium crinale.

Contactless approach through portable vibrational spectroscopy to identify the binders of German and Italian Modern Paintings

Order variations along silkworm/spider silk

Talanta, 2021

International Journal of Biological Macromolecules, 2020

Zoomorphology, 2020

European Journal of Organic Chemistry, 2018

Journal of Composite Materials, 2017

Journal of Raman Spectroscopy, 2016

Journal of structural biology, Jan 6, 2016

Journal of Proteomics, 2017

Journal of Polymer Science Part B: Polymer Physics, 2000

Design and characterization of anchoring amphiphilic peptides and their interactions with lipid vesicles

Biopolymers, 1999

Biophysical Journal, 2001

Bioconjugate Chemistry, 2000

Annals of the New York Academy of Sciences

Calcium Deposits in the Crayfish, Cherax quadricarinatus: Microstructure Versus Elemental Distribution

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada, Jan 28, 2016

Acta Pharmaceutica Sciencia

The ‘best’ silk fibres (natural polyamide), present a very high breaking resistance but also a hu... more The ‘best’ silk fibres (natural polyamide), present a very high breaking resistance but also a huge variability (typically 200-1000 MPa). Films made of Bombyx mori fibroin showed good biocompatibility suitable for biomedical applications, as silk was used as suture material for centuries. Solubilisation process degrades macromolecules and mechanical properties of regenerated silk, ultimate strain and stress, are drastically lower (a few %) than those measured for raw fibres. The goal of this work is to improve tensile mechanical properties of regenerated silk films by silk fibres reinforcement, degummed or not. Degummed fibres (i.e. without sericin coating) are dissolved and dialysed in order to obtain a liquid/gel more or less viscous followed by evaporation to obtain silk matrix. Structure and orientation modifications of fibroin macromolecules are monitored by Raman spectroscopy. The incorporation of a few fibre % vol improved the breaking resistance and dissipation. The mechanic...