Mechanical anisotropy of hair affected by genetic diseases highlights structural information related to differential crosslinking in keratins.

Previous work with Atomic Force Microscope (AFM) nanoindentation, on longitudinal and cross-sections of the human hair fibre, allowed for the derivation of a model for the mechanical behaviour of human hair, called the Anisotropic Index. Expanding that research further, and by applying this model, the nanomechanical behaviour of hairs from patients with the disease Trichothiodystrophy (TTD) has been examined and structural insights, gained from combining the AFM results with Differential Scanning Calorimetry (DSC) experiments and tensile measurements, suggests that TTD-affected hairs have a relatively increased amount of Keratin Intermediate Filaments, contained in compartments of differing crosslinking extent. The associated calculations of axial and transverse Young's Moduli deliver values in good agreement with the measured fibre mechanics. Furthermore, comparing these findings with the results previously obtained from the study of hairs from patients with the disease Monilethrix, it is shown that the Anisotropic Index correlates well with the known deficiencies in both hair types obtained from such patients and allows for discerning between the Control hair and from those affected by the two diseases. AFM nanoindentation along and across the fibre axis and the Anisotropic Index thus appear to reveal structural details of hair not otherwise acquirable, whilst DSC may offer a quick and simple method for distinguishing between different severities of TTD.

Learning from hair moisture sorption and hysteresis.

OBJECTIVE: The process of moisture sorption and desorption by human hair was analysed for extracting hints on the hair structure. METHODS: The isotherms of moisture sorption and desorption by hair were recorded for untreated and chemically treated (permed and bleached) hair. Data of swelling were also considered. RESULTS: By examining the swelling and moisture sorption of keratin fibres, it is possible to conclude that hysteresis is quite improbably caused by capillary condensation. The mobility of the protein chains and the strength of the bonds binding water molecules to the active sites inside the matrix are proposed as causes instead. The concept of "breaking symmetry", derived from moisture sorption- desorption data, and the method of evaluating this parameter, is proposed as a way of characterizing the chemical treatment of hair. The results show that bleaching produces a larger breaking of symmetry than perming, and this is suggested to be due to new hydrogen bonds, created as a result of the chemical treatment, replacing the original disulphide bonds, which are of different strength compared to the bonds of untreated hair. The quantitative sorption data matched well to the model of grains of matrix enveloped in layers of water molecules at increasing relative humidity, up to 100%. The analysis suggested that, aside from the glass transition event occurring at around 60%-70% relative humidity, there is another, less examined, transition occurring at around 30% relative humidity, assigned to the opening of the hair inner structure, and accommodation of more water molecules. Both transitions are reflected by corresponding changes in the fibre mechanical behaviour. CONCLUSION: The moisture sorption-desorption by hair was shown to not only allow a quantitative differentiation among various cosmetic treatments of the hair but to also provide valuable information on the structure of the fibre.

OBJECTIF: Le processus de sorption et de désorption de l'humidité par le cheveu humain a été analysé pour en extraire des informations sur la structure du cheveu. MÉTHODES: Les isothermes de sorption et de désorption de l'humidité par les cheveux ont été enregistrées pour des cheveux non traités et traités chimiquement (permanente et blanchie). Les données de gonflement ont également été prises en compte. RÉSULTATS: En examinant le gonflement et la sorption de l'humidité des fibres de kératine, il est possible de conclure que l'hystérésis est très improbablement causée par la condensation capillaire. La mobilité des chaînes de protéines et la force des liaisons liant les molécules d'eau aux sites actifs à l'intérieur de la matrice sont plutôt proposées comme causes. Le concept de "rupture de symétrie", dérivé des données de sorption-désorption de l'humidité, et la méthode d'évaluation de ce paramètre, sont proposés comme moyen de caractériser le traitement chimique des cheveux. Les résultats montrent que la blanchiment produit une rupture de symétrie plus importante que la permanente, ce qui serait dû à de nouvelles liaisons hydrogène, créées à la suite du traitement chimique, remplaçant les liaisons disulfure d'origine, qui sont de force différente par rapport aux liaisons des cheveux non traités. Les données quantitatives de sorption correspondent bien au modèle des grains de la matrice enveloppés dans des couches de molécules d'eau à une humidité relative croissante, jusqu'à 100 %. L'analyse a suggéré qu'à part la transition vitreuse qui se produit à environ 60-70 % d'humidité relative, il y a une autre transition, moins examinée, qui se produit à environ 30 % d'humidité relative, attribuée à l'ouverture de la structure interne du cheveu et à l'accommodation de plus de molécules d'eau. Les deux transitions sont reflétées par des changements correspondants dans le comportement mécanique de la fibre. CONCLUSION: La sorption-désorption de l'humidité par le cheveu permet non seulement de différencier quantitativement les différents traitements cosmétiques du cheveu, mais aussi de fournir des informations précieuses sur la structure de la fibre.

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress.

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central "rod" region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.

Cuticle - Designed by nature for the sake of the hair.

OBJECTIVE: The cuticle of human hair has been examined, via a range of analytical methods, in order to reveal previously unknown information about its structure and to deepen understanding of its contribution to fibre properties. METHODS: Cross-sections of hair fibre have been examined with X-ray microdiffraction oriented perpendicular to the surface of the cross-sections. AFM investigations were carried out for further investigating and deciphering the structure of the cuticle. Moisture sorption analytics of cuticle separated from fibre and mechanical tests of decuticled fibres against virgin fibres were used for understanding the role of the cuticle in the economy of hair fibre. RESULTS: Previously unknown swelling behaviour of the hair cuticle during moisture sorption has been revealed, as has an increased significance of the cuticle's role in moisture management at higher values of relative humidity. Through AFM investigation, the reaction of hair cuticles with chlorine water has further strengthened the idea that the Allwörden membrane does not exist, and is actually an artefact of the delamination of the A-layer and exocuticle from the underlying endocuticle. Using decuticled fibres for stress-strain tests, and by comparing the results with those of virgin fibres, the effect of the cuticle on the post-yield area of the hair fibre stress-strain diagram has also been demonstrated. Finally, X-ray microdiffraction and AFM investigations suggest that the cuticle possesses a small-scale ordered structure, based on possibly not fully crystalline and irregularly arranged α-helices oriented almost perpendicular to the growth axis of the fibre and enhancing the general description of cuticle as the protective layer of the fibre. CONCLUSION: The role of the cuticle for the hair fibre is more complex than previously thought. The cuticle is demonstrated not only to possess a hidden rod-matrix structure, that supports its protective nature, but also to play specific roles in the fibre's response to moisture, and in fibre mechanical behaviour.

OBJECTIF : la cuticule des cheveux humains a été examinée à l'aide d'un ensemble de méthodes analytiques, afin de révéler des informations jusqu'alors inconnues sur sa structure et d'approfondir la compréhension de son rôle dans les propriétés de la fibre. MÉTHODES : des coupes transversales de fibres capillaires ont été examinées par microdiffraction radiographique orientée perpendiculairement à la surface des coupes. Des expérimentations par AFM ont été réalisées pour approfondir les recherches et découvrir la structure de la cuticule. Des analyses d'absorption de l'humidité de la cuticule séparée de la fibre et des tests mécaniques des fibres décuticulées par rapport aux fibres vierges ont été utilisés pour comprendre le rôle de la cuticule dans la préservation de la fibre capillaire. RÉSULTATS : un comportement de gonflement jusqu'alors inconnu de la cuticule des cheveux durant l'absorption de l'humidité a été révélé, de même qu'une importance accrue du rôle de la cuticule dans la gestion de l'humidité à des valeurs plus élevées d'humidité relative. À l'aide des expérimentations par AFM, la réaction de la cuticule des cheveux avec de l'eau chlorée a à nouveau renforcé l'idée selon laquelle la membrane d'Allwörden n'existe pas et est en réalité un artéfact de délaminage de la couche A et de l'exocuticule provenant de l'endocuticule sous-jacente. L'utilisation de fibres décuticulées pour des tests de contrainte et la comparaison des résultats avec ceux de fibres vierges ont également démontré l'effet de la cuticule sur la zone post-rendement du diagramme de contrainte de la fibre capillaire. Enfin, les expérimentations par microdiffraction radiographique et AFM suggèrent que la cuticule possède une structure ordonnée à petite échelle, basée sur des hélices alpha potentiellement non entièrement cristallines et disposées de manière irrégulière, orientées presque perpendiculairement à l'axe de croissance de la fibre, améliorant la description générale de la cuticule comme couche protectrice de la fibre. CONCLUSION : le rôle de la cuticule pour la fibre capillaire est plus complexe qu'on ne le pensait. Il a été démontré que la cuticule possède non seulement une structure rod-matrix cachée, qui maintient sa nature protectrice, mais joue également des rôles spécifiques dans la réponse de la fibre à l'humidité et dans son comportement mécanique.

Nanomechanical properties of Monilethrix affected hair are independent of phenotype.

Utilising the AFM nanoindentation technique for the study of hair cross- and longitudinal sections, the mechanical anisotropy of human hair fibres affected by a rare congenital condition, Monilethrix, has been investigated for the first time. Supported by X-ray microdiffraction data, and applying a model based on an ideal composite material consisting of rods (KIFs) and matrix (KAPs) to Monilethrix affected fibres, it has been shown that the results could be grouped into clearly different classes, namely: almost isotropic behaviour for Monilethrix affected hairs and anisotropic behaviour for Control hair. Moreover, AFM nanoindentation of hair cross sections has demonstrated, also for the first time that hairs affected by Monilethrix have a continuous, and not periodic, weakness within the cortex. This has been attributed to disruptions in the KIF-KIF, KIF-intermacrofibrillar matrix or KIF-desmosome complexes within the hair shaft, as suggested by X-ray microdiffraction examination. Hairs from a patient exhibiting no obvious phenotype exhibited similar mechanical weakness despite the otherwise normal visual appearance of the fibre. This further supports a hypothesis that the beaded appearance of Monilethrix hair is a secondary factor, unrelated to the inherent structural weakness.

Influence of composition on the thermal behavior of oils extracted from the seeds of some Romanian grapes.

BACKGROUND: The oils obtained from grape seeds are becoming a valuable way of exploiting winery waste and their properties are scrutinized for evaluating the potential usages. We examined the oils extracted with petroleum ether from grape seeds of four varieties of grapes (Cabernet Sauvignon, Feteasca Neagra, Merlot and Pinot Noir) from Romania looking at the influence of the fatty acid profile and of the antioxidants on their thermal behavior. RESULTS: The fatty acid profiles of the oils were evaluated by 1 H-NMR spectroscopy, and the oil antioxidant capacity was determined by cupric ion reducing antioxidant capacity (CUPRAC) method. The main fatty acid component in all the oils is linoleic acid (over 70%), which, due to its known health benefits, make the oils of commercial interest. The thermal stability of grape seed oils appears to be mainly influenced by the percentage of polyunsaturated fatty acids in their composition, less polyunsaturated fatty acids making the oils more stable. The antioxidant compounds affect only the initial stage of the decomposition by limiting the formation of hydroperoxides in the allylic positions of the fatty acid chain. CONCLUSION: Compared to pure samples of glyceryl-unsaturated fatty acids (glyceryl-trioleate, glyceryl-trilinoleate), the grape seed oils exhibit higher thermal stability, due to the presence of antioxidant compounds and to a synergistic action of unsaturated and saturated fatty acids, smaller percentage of the polyunsaturated and higher percentage of the saturated fatty acids enhancing the stability. © 2019 Society of Chemical Industry.

Relevance and Evaluation of Hydrogen and Disulfide Bond Contribution to the Mechanics of Hard α-Keratin Fibers.

We propose a simple mechano-chemical model for the dependence of Young's modulus of α-keratin fibers on the hydrogen and disulfide bonds existing in the matrix and evaluate the relative change in bonding following an oxidative chemical treatment. Atomic force microscopy nanoindentation of longitudinal and cross sections of the fiber showed that, although the oxidative treatment breaks a significant amount of disulfide bonds, it introduces compensatory hydrogen bonds that maintain fiber elasticity at values comparable with those of the untreated fiber under dry conditions. The striking influence of humidity on the hydrogen bonding in keratin fibers is also evaluated. The hydrogen bonds are labeled as "type 1" and "type 2" hydrogen bonds; newly formed hydrogen bonds, type 2, are more labile than those native (type 1) to the matrix. Examining their contribution to Young's modulus of the matrix allowed for quantifying the loss of disulfide bonds, and the result matched the decrease in cystine, measured by amino-acid analysis, caused by the oxidative treatment.

The Thermodynamics of Trichocyte Keratins.

This chapter is an attempt at an excursion into the world of keratins with the help of thermodynamics.After briefly introducing some of the thermodynamic concepts involved in deciphering the behaviour of keratins, we will use them to look into the process of aggregation of keratin molecules into intermediate filaments, and keratin fibres, and then for analysing how keratin materials react to mechanical, thermal and moisture stresses, respectively.In most of the cases entropy appears to be the major driving force of the response occurring in keratins under environmental assault. This fact points to the important role played for keratins by temperature, which, aside from influencing the kinetics of the processes (accelerating or decelerating the rates of the rates), helps increase or decrease the entropic contribution to the Gibbs free energy and, thus, allows thermodynamically the occurrence of the observed behaviour of keratins.

Hair Mechanical Anisotropy-What Does It Tell Us?

Hair fibers were examined by atomic force microscopy, nanoindentation. By indenting along (longitudinal) and across (transversal) the fiber, we evaluated the Young's modulus and its dependence on the moisture content (relative humidity) of the environment. The ratio of the two values collected for Young's modulus, at a given relative humidity, is defined as the anisotropy index (IA) of the fiber and the acquired results give the evolution of the index of anisotropy with the relative humidity. The use of the model of composite materials allowed us to relate the anisotropy index to the fiber internal architecture. The evaluation of the results acquired on the components of the fiber, within the frame of this model, ultimately points to a possible micro-structure of exocuticle, hindered under usual circumstances by its heavy cross-linking and only noticeable when the absorbed moisture swells the surrounding network and annuls, in this way, its effect.

The structure of the "amorphous" matrix of keratins.

Various keratin fibers, particularly human hairs, were investigated by transmission electron microscopy, TEM, solid-state 1H NMR and Transient Electro-Thermal Technique, TET. The results converge to suggest that the matrix of keratin fiber cortex, far from being amorphous, has a well-defined nano-scale grainy structure, the size of these grains being around 2-4nm. The size of the grains appears to strongly depend on the chemical treatment of the fiber, on the temperature and on the relative humidity of the environment, as well as on the physiological factors at the level of fiber production in follicle. By suggesting an organization at the nano-scale of the protein chains in these grains, likely to be Keratin Associated Proteins, the results challenge the view of matrix as a homogeneous glassy material. Moreover, they indicate the potential of further investigating the purpose of this structure that appears to reflect not only chemical treatments of keratins but also biological processes at the level of the follicle.

Synthesis and Characterization of Methyl Cellulose/Keratin Hydrolysate Composite Membranes.

It is known that aqueous keratin hydrolysate solutions can be produced from feathers using superheated water as solvent. This method is optimized in this study by varying the time and temperature of the heat treatment in order to obtain a high solute content in the solution. With the dissolved polypeptides, films are produced using methyl cellulose as supporting material. Thereby, novel composite membranes are produced from bio-waste. It is expected that these materials exhibit both protein and polysaccharide properties. The influence of the embedded keratin hydrolysates on the methyl cellulose structure is investigated using Fourier transform infrared spectroscopy (FTIR) and wide angle X-ray diffraction (WAXD). Adsorption peaks of both components are present in the spectra of the membranes, while the X-ray analysis shows that the polypeptides are incorporated into the semi-crystalline methyl cellulose structure. This behavior significantly influences the mechanical properties of the composite films as is shown by tensile tests. Since further processing steps, e.g., crosslinking, may involve a heat treatment, thermogravimetric analysis (TGA) is applied to obtain information on the thermal stability of the composite materials.

Keratin made micro-tubes: The paradoxical thermal behavior of cortex and cuticle.

Keratin micro-tubes were obtained by heating medullated keratin fibres to temperatures above 230°C under nitrogen atmosphere, when, as documented by microscopy, the cortex (the core of the fibre) melts from the medulla outwards, followed by pyrolysis of the material through the remaining solid cuticle (shell) layer. The resulted hollow tubes from fibres void of cortical material keep the external cuticle structure, as shown by AFM investigation, and the moisture sorption properties of the initial keratin fibre. Despite similar amino-acid compositions of cuticle and cortex the two morphological components differ significantly in their thermal behaviour, which appears to be a "cortex-cuticle thermal stability paradox".

Chapter 4. Cytomechanics of hair basics of the mechanical stability.

Hair is a complex "cornified" multicellular tissue composed of cuticle and cortex cells mechanically acting as a whole. The cuticle cells overlap and cortex cells interdigitate, all cells being composed of different morphological elements and separated by the cell membrane complex (CMC). The CMC and the morphological elements of the cortex cells, the macrofibrils, composed of microfibrils or intermediate filaments (IFs), and the intermacrofibrillar and intermicrofibrillar cement or the amorphous matrix material determine the mechanical properties of hair. The IFs consist of alpha-keratin molecules being arranged in a sophisticated way of two parallel monomers and antiparallel and shifted dimers rationalized by the amino acid composition and sequence. The mechanical properties of hair result from mechanical interlocking effects, hydrophobic effects, hydrogen bridges, Coulombic interactions, and (covalent) isodipeptide and, in particular, disulfide bridges on a molecular level. The mechanical models applied to hair are based on a simple two-component system, the microfibril/matrix structure. An important regime of the stress-strain curve is the transition of the molecules of the microfibrils from the alpha-helical to the beta-sheet structure. Due to the longitudinal orientation of the IF molecules the longitudinal swelling of the fibers in water is negligible, the radial swelling, however, is substantial.

Morphology and molecular mobility of fibrous hard alpha-keratins by 1H, 13C, and 129Xe NMR.

The morphology and molecular mobility changes of the side chains for hard alpha-keratin due to oxidative and reductive/oxidative treatments for temperatures around the DSC denaturation peak were investigated by (1)H, (13)C, and (129)Xe NMR spectroscopy and (1)H spin diffusion. Proton wide-line spectra were used to obtain the phase composition (rigid, interface, and amorphous fractions) and molecular dynamics of each phase. Proton spin diffusion experiments using a double-quantum filter and initial rate approximation were employed to obtain the dependence of the rigid domain sizes on chemical treatments and denaturation temperatures. A drastic reduction in the rigid domain thickness takes place for the reductive/oxidative treatment. The keratin mobility gradient in the interfacial region at different denaturation temperatures was measured for hard alpha-keratin from (1)H spin diffusion data. (13)C CPMAS spectra were used to provide a detailed examination of the effects of the chemical treatments especially on the disulfide bonds. Thermally polarized (129)Xe spectra suggest the existence of voids in the hard alpha-keratin induced by the reductive and oxidative treatment. The surface of the hard alpha-keratin fiber surface is probed by the laser hyperpolarized (129)Xe. A qualitative model describing the changes induced in hard alpha-keratin protein by chemical transformation was developed and could be correlated with the changes in domain thickness, phase composition, and molecular dynamics.

Non-isothermal kinetics of hard alpha-keratin thermal denaturation.

DSC measurements carried out at different heating rates were used for the kinetic analysis of the endothermic process assigned to the denaturation of the helical material from human hair in water excess. We found that the kinetic mechanism is autocatalytic and that the value of the activation energy is close to disulphide bond scission rather than to protein denaturation. This allowed us to propose a multistep mechanism for the thermal denaturation of hard alpha-keratins in water excess that relies on the 3-phase model which describes their structure. The limiting step of the thermal denaturation process is then the scission of S-S bonds between the main morphological components, namely IF and matrix (IFAP). The theoretical proposed model shows a good agreement with the experimental recorded data.

Thermal denaturation of hydrated wool keratin by 1H solid-state NMR.

Thermal denaturation of hydrated keratin in wool was investigated by NMR using 1H wide-line spectra to obtain the phase composition and 1H spin-diffusion experiments using a double-quantum filter to obtain the domain sizes for the wool fibers. The denaturation process detected by DSC takes place for wool fibers in deuterated water in the temperature range 140-144 degreeC. The phase composition measured by 1H wide line NMR spectra reveals a rigid, semirigid and an amorphous phase for temperatures in the range 25-160 degreeC. A dramatic change in the phase composition was detected around 142 degreeC, corresponding to the denaturation temperature. The morphological domain sizes measured by 1H spin-diffusion NMR experiments were obtain from the solutions of the spin-diffusion equations for two-dimensional rectangular and cylindrical morphologies. The keratin mobility gradient in the interfacial region at different denaturation temperatures was measured from the 1H spin-diffusion data. A qualitative model describing the denaturation process of hydrated keratin protein was developed that explains the changes in domain thickness, spin diffusivities, phase composition, and thermodynamic parameters.

Hair--the most sophisticated biological composite material.

Hair is a proteinaceous fibre with a strongly hierarchical organization of subunits, from the alpha-keratin chains, via intermediate filaments, to the fibre. The chemistry of the different morphological compartments results in exciting physical properties, including the hydrophilic/hydrophobic paradox. The present tutorial review will be of interest for protein- as well as polymer chemists, who want to learn from nature, and also for biochemists interested in the cytoskeleton and particularly in intermediate filaments; it also presents a scientific basis for hair cosmetics.

Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water.

Keratins are self-organized proteins that are abundantly available in wool, feather, human hair, etc., making them a potential cheap feedstock for the modification of amino acids. This paper explores the hydrolysis of keratin in water under specific pressure-temperature conditions where the hydrolysis through scission of the protein chain yields oligopeptides. Here we report for the first time that, under appropriate conditions, these oligopeptides self-assemble into a hierarchical architecture, the process being followed in time by optical microscopy. Birefringent needle-like crystals are observed which tend to nucleate heterogeneously. When given sufficient time, these needles become tens of microns in length and act as further nuclei, developing a highly repetitive structure of several hundreds of microns in size. Micro-focus X-ray diffraction studies supported by in situ microscopy reveal that these needles have a crystal structure similar to that of the native protein, although better organized along the ab-plane. Spectroscopic studies on these structures show crystalline bands that disappear above 150 degrees C, coinciding with an endothermic peak in DSC. Amino acid analysis shows that the self-assembled birefringent entities are indeed oligopeptides, consisting of sequences of approximately 40 amino acids. The proposed ecofriendly route provides an effective route for obtaining oligopeptides that can be used as important building blocks for the synthesis of a range of novel polymers. The oligopeptides obtained from the sustainable source can be used as important building blocks for the synthesis of a range of novel polymers.