Analysis of structural changes in bleached keratin fibers (black and white human hair) using Raman spectroscopy

To investigate the influence of bleaching treatments on keratin fibers, the structure of cross-sections at various depths of bleached human hair (black and white human hair) was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. The S-S band intensity existing from the cuticle region to the center of cortex region of virgin white human hair decreased, while the S-O band intensity at 1040 cm(-1), assigned to cysteic acid, increased by performing the bleaching treatment. Especially, the S-O band intensity of the cuticle region increased remarkably compared with that of the cortex region. Also, the amide III (unordered) band intensity in the cortex region increased, indicating that some of the proteins existing throughout the cortex region changed to the random coil form. Moreover, it has been found that the S-S band intensity existing from the cuticle region to the center of the cortex region of the virgin black human hair decreased remarkably, while the S-O band intensity increased significantly compared with that of the virgin white human hair by performing the bleaching treatment. From these experiments, we concluded that the melanin granules including metal ions act as a decomposition accelerator for the oxidizing agent, thereby leading to a higher level of disulfide (-SS-) group cleavage in the black human hair compared with that of the white human hair.     

In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy

The biophysical and biomechanical properties of the crystalline lens (e.g., viscoelasticity) have long been implicated in accommodation and vision problems, such as presbyopia and cataracts. However, it has been difficult to measure such parameters noninvasively. Here, we used in vivo Brillouin optical microscopy to characterize material acoustic properties at GHz frequency and measure the longitudinal elastic moduli of lenses. We obtained three-dimensional elasticity maps of the lenses in live mice, which showed biomechanical heterogeneity in the cortex and nucleus of the lens with high spatial resolution. An in vivo longitudinal study of mice over a period of 2 months revealed a marked age-related stiffening of the lens nucleus. We found remarkably good correlation (log-log linear) between the Brillouin elastic modulus and the Young's modulus measured by conventional mechanical techniques at low frequencies (~1 Hz). Our results suggest that Brillouin microscopy is potentially useful for basic and animal research and clinical ophthalmology.     

Distinguishing Central African rodents and shrews using their hair morphology

Species identification methods are needed for small mammals. Herein, we present a simple and efficient identification key based on the hair morphology of Central African rodents (Rodentia) and shrews (Soricomorpha). A total of 1,320 museum hair samples from 51 species were analysed with an optical light microscope. One‐third (31.37%) of these samples could be identified to the species level using four morphological characters (medulla, cuticle, size and colour). The remaining species formed nine groups of two to 10 species each which could not be discriminated because of overlapping characteristics between different taxa. In addition, shrew, dormice and squirrel hair samples were clearly distinguishable from the other samples because they were either shorter (0.68 ± 0.19 cm) or longer (1.86 ± 0.3 and 1.85 ± 0.63 cm), respectively. In addition, 19 (43.18%) field‐collected samples of unknown origin were successfully identified to the level of species or group of species. Thus, to increase the efficiency of this identification key, the inclusion of more morphological characteristics (i.e. hair diameter and shape, cuticle index, cortex types) and DNA barcoding should be considered. Finally, the proposed identification key could be used as a simple and efficient tool for species inventories and ecological studies of targeted taxa in Central Africa.     

Nanoscale strain-hardening of keratin fibres

Mammalian appendages such as hair, quill and wool have a unique structure composed of a cuticle, a cortex and a medulla. The cortex, responsible for the mechanical properties of the fibers, is an assemblage of spindle-shaped keratinized cells bound together by a lipid/protein sandwich called the cell membrane complex. Each cell is itself an assembly of macrofibrils around 300 nm in diameter that are paracrystalline arrays of keratin intermediate filaments embedded in a sulfur-rich protein matrix. Each macrofibril is also attached to its neighbors by a cell membrane complex. In this study, we combined atomic force microscopy based nano-indentation with peak-force imaging to study the nanomechanical properties of macrofibrils perpendicular to their axis. For indentation depths in the 200 to 500 nm range we observed a decrease of the dynamic elastic modulus at 1 Hz with increasing depth. This yielded an estimate of 1.6GPa for the lateral modulus at 1 Hz of porcupine quill's macrofibrils. Using the same data we also estimated the dynamic elastic modulus at 1 Hz of the cell membrane complex surrounding each macrofibril, i.e., 13GPa. A similar estimate was obtained independently through elastic maps of the macrofibrils surface obtained in peak-force mode at 1 kHz. Furthermore, the macrofibrillar texture of the cortical cells was clearly identified on the elasticity maps, with the boundaries between macrofibrils being 40-50% stiffer than the macrofibrils themselves. Elasticity maps after indentation also revealed a local increase in dynamic elastic modulus over time indicative of a relaxation induced strain hardening that could be explained in term of a α-helix to β-sheet transition within the macrofibrils.     

Cuticular microstructures turn specular black into matt black in a stick insect

The stick insect Peruphasma schultei stands out from other insects by its deep matt black cuticle. We tested whether the appearance of P. schultei is due to microstructures of the cuticle, a phenomenon that has recently been described for the velvet black scales of the Gaboon viper. The shiny black stick insect Anisomorpha paromalus served as a control. We found that the P. schultei cuticle is characterised by two different types of microstructures, tall elevations with a maximum size of 18 μm and small structures with a height of 4 μm. Other than in the snake, P. schultei microstructures do not bear nanostructures. The microstructures scatter light independently of the viewing angle. This causes the matt appearance of the cuticle, whereas pigments are responsible for the black colouration, resulting in a maximum reflectance of 2.8% percent. The microstructures also cause the hydrophobic properties of the cuticle with contact angles near 130°. Resin replicas and bleaching of the cuticle strongly support these results. Moreover, the matt black cuticle has a higher heat absorption compared to the control. We discuss the selective benefit of the matt black appearance of P. schultei in the context of behaviour, ecology and phylogeny.     

Viscoelastic properties' characterization of corneal stromal models using non-contact surface acoustic wave optical coherence elastography (SAW-OCE)

Viscoelastic characterization of the tissue-engineered corneal stromal model is important for our understanding of the cell behaviors in the pathophysiologic altered corneal extracellular matrix (ECM). The effects of the interactions between stromal cells and different ECM characteristics on the viscoelastic properties during an 11-day culture period were explored. Collagen-based hydrogels seeded with keratocytes were used to replicate human corneal stroma. Keratocytes were seeded at 8 × 10³ cells per hydrogel and with collagen concentrations of 3, 5 and 7 mg/ml. Air-pulse-based surface acoustic wave optical coherence elastography (SAW-OCE) was employed to monitor the changes in the hydrogels' dimensions and viscoelasticity over the culture period. The results showed the elastic modulus increased by 111%, 56% and 6%, and viscosity increased by 357%, 210% and 25% in the 3, 5 and 7 mg/ml hydrogels, respectively. To explain the SAW-OCE results, scanning electron microscope was also performed. The results confirmed the increase in elastic modulus and viscosity of the hydrogels, respectively, arose from increased fiber density and force-dependent unbinding of bonds between collagen fibers. This study reveals the influence of cell-matrix interactions on the viscoelastic properties of corneal stromal models and can provide quantitative guidance for mechanobiological investigations which require collagen ECM with tuneable viscoelastic properties.     

Worms under Pressure: Bulk Mechanical Properties of C. elegans Are Independent of the Cuticle

The mechanical properties of cells and tissues play a well-known role in physiology and disease. The model organism Caenorhabditis elegans exhibits mechanical properties that are still poorly understood, but are thought to be dominated by its collagen-rich outer cuticle. To our knowledge, we use a novel microfluidic technique to reveal that the worm responds linearly to low applied hydrostatic stress, exhibiting a volumetric compression with a bulk modulus, κ = 140 ± 20 kPa; applying negative pressures leads to volumetric expansion of the worm, with a similar bulk modulus. Surprisingly, however, we find that a variety of collagen mutants and pharmacological perturbations targeting the cuticle do not impact the bulk modulus. Moreover, the worm exhibits dramatic stiffening at higher stresses—behavior that is also independent of the cuticle. The stress-strain curves for all conditions can be scaled onto a master equation, suggesting that C. elegans exhibits a universal elastic response dominated by the mechanics of pressurized internal organs.     

Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles

The major histological components of the hair follicle are the hair cortex and cuticle. The hair cuticle cells encase and protect the cortex and undergo a different developmental program to that of the cortex. We report the molecular characterization of a set of evolutionarily conserved hair genes which are transcribed in the hair cuticle late in follicle development. Two genes were isolated and characterized, one expressed in the human follicle and one in the sheep follicle. Each gene encodes a small protein of 16 kD, containing greater than 50 cysteine residues, ranging from 31 to 36 mol% cysteine. Their high cysteine content and in vitro expression data identify them as ultra-high-sulfur (UHS) keratin proteins. The predicted proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Genomic blots reveal that the UHS keratin proteins are encoded by related multigene families in both the human and sheep genomes. Tissue in situ hybridization demonstrates that the expression of both genes is localized to the hair fiber cuticle and occurs at a late stage in fiber morphogenesis.     

Protein structural changes in keratin fibers induced by chemical modification using 2-iminothiolane hydrochloride: a Raman spectroscopic investigation

For the purpose of investigating in detail the influence of chemical modification using 2-iminothiolane hydrochloride (2-IT) on keratin fibers, the structure of cross-sections at various depths of white human hair, treated with 2-IT and then oxidized, was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. In particular, the beta-sheet and/or random coil content (beta/R) and the alpha-helix (alpha) content in human hair fibers were estimated by amide I band analysis. The S-S band intensity, amide III (unordered) band intensity, and beta/R content existing from the cuticle region to the center of cortex region of virgin white human hair remarkably increased by performing the chemical modification using 2-IT. On the other hand, not only the S-S band intensity, but also S-O band intensity existing throughout the cortex region of the bleached (damaged) white human hair increased by performing chemical modification using 2-IT. In particular, beta/R content existing throughout the cortex region of the bleached white human hair decreased, while the skeletal C-C stretch (alpha) band intensity at 935 cm(-1) and the alpha content remarkably increased. This indicates a secondary structural change from the random coil form to the alpha-helix form in the proteins existing throughout the cortex region. From these experiments, we concluded that the formation of new disulfide (-SS-) groups resulting from chemical modification using 2-IT induced the secondary structural changes of proteins existing throughout the cortex region.     

Soybean Hull Insoluble Polysaccharides: Improvements of Its Physicochemical Properties Through High Pressure Homogenization

Soybean hull is an agroindustrial waste which has not been fully studied as a food ingredient. The aims of this work were to obtain insoluble fibers from soybean hull and to evaluate the effect of high pressure homogenization (HPH) on its physicochemical properties. Hull insoluble polysaccharides (HIPS) were obtained in a single step, as the insoluble residue after pectin removal. FTIR showed bands corresponding to cellulose and hemicellulose in HIPS, and thermogravimetric analysis showed two degradation events at 236.3 °C and 325.6 °C, corresponding to cellulose and hemicellulose, respectively. HIPS dispersions (pH 3.00) were subjected to HPH by three cycles at increasing pressures (up to 1000 bar), obtaining soybean hull nanofibers. SEM images show that HPH at 1000 bar reduced the dimensions of the fiber bundle from 30 to 90 μm in length and 9–15 μm in diameter to nanofibers of 10–30 μm in length and 100–400 nm in diameter. AFM further confirms a heterogeneous distribution of sizes in HIPS₈₀₀ and HIPS₁₀₀₀, evidencing the presence of individual nanofibers with diameters around 50 ± 10 nm and 40 ± 10 nm, respectively, with several μm in length. Furthermore, an increase in water holding capacity from 2.1 to 61 g_water/g_{dry matter} and viscosity from 0.39 to 34,945 Pa.s were achieved as HPH at 1000 bar treatment was applied. HPH increased the interfacial area and promoted the interconnection of fibers in a hydrated gel-like structure. This explains flow behavior, which was extensively studied in this work: three-region viscosity profile (shear-thinning, plateau or shear-thickening and shear-thinning) and a pronounced hysteresis loop. Oscillatory rheology was used to study the viscoelastic behavior of HIPS dispersions. HIPS are a source of nanofibers, easy to obtain through a single step of chemical treatment followed by the application of high pressures. It is remarkable that the use of few chemical solvents is favorable from an environmental point of view. This work also suggests a potential application of HIPS to improve physicochemical and structural properties in acidic foods.

Graphical Abstract

     

Effect of sample treatment on biomechanical properties of insect cuticle

Experimental limitations often prevent to perform biomechanical measurements on fresh arthropod cuticle samples. Hence, in many cases short- or long-term storage of samples is required. So far, it is not known whether any of the standard lab-techniques commonly used to fix or store insect cuticle samples in any way affects the biomechanical properties of the respective samples.In this paper we systematically address this question for the first time, with a focus on practical, easily accessible and common lab-methods including storage in water, ethanol, glutaraldehyde, freezing and desiccation. We performed a comprehensive and sensitive non-destructive Dynamic Mechanical Analysis (DMA) on locust hind leg tibiae using a three-point-bending setup. Our results show that from all tested treatments, freezing samples at −20 °C was the best option to maintain the original values for Young's modulus and damping properties of insect cuticle. In addition, our results indicate that the damping properties of locust hind legs might be mechanically optimized in respect to the jumping and kicking direction.     

Analysis of structural changes in permanent waved human hair using Raman spectroscopy

To investigate the mechanism leading to the reduction in tensile strength of permanent waved human hair, the structure of cross-sections at various depths of permanent waved white human hair was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. The beta-sheet and/or random coil content (beta/R) and the Amide III(unordered) band intensity existing throughout the cortex region of virgin white human hair remarkably increased, while the alpha-helix (alpha) content slightly decreased by performing the permanent waving treatment. This suggests a secondary structural change from the alpha-helix form to the random coil form in the proteins existing in the microfibril of the cortex region. On the other hand, the S-S band intensity existing in the matrix of the cortex region almost did not change, despite the reduction in the tensile strength of the white human hair following the permanent waving treatment. Moreover, the transmission electron microscope observation shows that the macrofibril (the microfibril and matrix) existing in the cortex region of the virgin white human hair was remarkably disturbed, while the cuticle region was almost unchanged by performing the permanent waving treatment. From these experiments, the authors concluded that some of proteins existing in the cortex region (the microfibril and matrix) of the virgin white human hair were changed, thereby leading to the remarkable reduction in the tensile strength of the white human hair after the permanent waving treatment.     

Age-associated thin hair displays molecular,structural and mechanical characteristic changes

Hair thinning occurs during normal chronological aging in women and in men leading to an increased level of thinner hair shafts alongside original thicker shafts. However, the characteristics of age-associated thin hairs remain largely unknown. Here we analyzed these characteristics by comparing at multiscale thin and thick hairs originated from Caucasian women older than 50 years. We observed that the cortex of thick hair contains many K35(+)/K38(?) keratinocytes that decrease in number with decreasing hair diameter. Accordingly, X-ray diffraction revealed differences supporting that thin and thick hairs are different with regards to the nature of the intermediate filaments making up their cortices. In addition, we observed a direct correlation between hair ellipticity and diameter with thin hairs having an unexpected round shape compared to the elliptic shape of thick hairs. We also observed fewer cuticle layers and a reduced frequency of a medullae in thin hairs. Regarding mechanical properties, thin hairs exhibited a surprising increased rigidity, a decrease of the viscosity and a decrease of the water diffusion coefficient. Hence, aged-associated thin hairs exhibit numerous modifications likely due to changes of hair differentiation program as evidenced by the modulations in the expression of hair keratins and keratin-associated proteins and by the X-ray diffraction specters. Hence, hair thinning with age does not consist simply of the production of a smaller hair. It is rather a more profound process likely relying on the implementation of an “aged hair program” that takes place within the hair follicle.     

Investigation of human hair cuticle structure by microdiffraction: direct observation of cell membrane complex swelling.

The cuticle of mammalian hair fibres protects the core of the fibre against physical and chemical stress. The structure and some of the properties of the cuticle have been extensively studied by electron microscopy. However, there is still a need for a less invasive structural probe. For this purpose, microdiffraction experiments have been carried out on human hair samples showing a characteristic small-angle X-ray scattering pattern for the cuticle. This pattern has been assigned to the cell membrane complex (CMC) between each cuticle scale. Using a simple model of the electron density within the CMC, values have been derived for the average thickness of the beta- and delta-layers which are close to those obtained by electron microscopy. In order to illustrate the potentialities of microdiffraction in studying the properties of the cuticle, the effect of water sorption has been monitored. Using the intensity modelling described above, a 10% swelling of the delta-layer's thickness has been observed. This study shows that structural modifications of the CMC by physical or chemical stress can be followed directly on the cuticle of human hair fibres by microdiffraction analysis.     

Analysis of structural change in keratin fibers resulting from chemical treatments using Raman spectroscopy

In order to investigate the influence of chemical treatments (reduction, heating, and oxidation) on keratin fibers, the structure of virgin white human hair resulting from a permanent hair straightening process at various depths of cross-sectional samples was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. The band shape of the cuticle was different from that of the cortex, and the cuticle had a more amorphous structure, compared with the cortex. The S-S band intensity existing in the hair surface remarkably decreased, while the S-S band intensity in the hair center was not changed by performing the reduction process. In the case of heating the keratin fibers after the reduction process, this tendency was unchanged. On the other hand, the amide III (unordered) band intensity in the cortex region increased, indicating that proteins existing throughout the cortex region caused a change to the random coil form. Moreover, approximately 95% of the disconnected -SS- groups were clearly reconnected by performing the oxidation process after heating (the degree of reconnection of -SS- groups was about 90%, in the case of oxidizing after reduction). From these experiments, we concluded that the heat treatment process in the permanent hair straightening treatment caused the randomization of proteins existing throughout the cortex region, thereby contributing to the acceleration of the reconnection of -SS- groups during the oxidation process.     

Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co- expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.     

Apparent elastic modulus and hysteresis of skeletal muscle cells throughout differentiation

The effect of differentiation on thetransverse mechanical properties of mammalian myocytes was determinedby using atomic force microscopy. The apparent elastic modulusincreased from 11.5 ± 1.3 kPa for undifferentiated myoblasts to45.3 ± 4.0 kPa after 8 days of differentiation (P < 0.05). The relative contribution of viscosity, as determined fromthe normalized hysteresis area, ranged from 0.13 ± 0.02 to0.21 ± 0.03 and did not change throughout differentiation. Myosinexpression correlated with the apparent elastic modulus, but neithermyosin nor -tubulin were associated with hysteresis. Microtubulesdid not affect mechanical properties because treatment with colchicinedid not alter the apparent elastic modulus or hysteresis. Treatmentwith cytochalasin D or 2,3-butanedione 2-monoxime led to a significantreduction in the apparent elastic modulus but no change in hysteresis.In summary, skeletal muscle cells exhibited viscoelastic behavior thatchanged during differentiation, yielding an increase in the transverseelastic modulus. Major contributors to changes in the transverseelastic modulus during differentiation were actin and myosin.

     

Three-dimensional architecture of macrofibrils in the human scalp hair cortex

Human scalp hairs are comprised of a central cortex enveloped by plate-like cuticle cells. The elongate cortex cells of mature fibres are composed primarily of macrofibrils-bundles of hard-keratin intermediate filaments (IFs) chemically cross-linked within a globular protein matrix. In wool, three cell types (ortho-, meso- and paracortex) contain macrofibrils with distinctly different filament arrangements and matrix fractions, but in human hair macrofibril-cell type relationships are less clear. Here we show that hair macrofibrils all have a similar matrix fraction (∼0.4) and are typically composed of a double-twist architecture in which a central IF is surrounded by concentric rings of tangentially-angled IFs. The defining parameter is the incremental angle increase (IF-increment) between IFs of successive rings. Unlike the wool orthocortex, hair double-twist macrofibrils have considerable inter-macrofibril variation in IF increment (0.05–0.35°/nm), and macrofibril size and IF increment are negatively correlated. Correspondingly, angular difference between central and outer-most IFs is up to 40° in small macrofibrils, but only 5–10° in large macrofibrils. Single cells were observed containing mixtures of macrofibrils with different diameters. These new observations advance our understanding of the nano-level and cell-level organisation of human hair, with implications for interpretation of structure with respect the potential roles of cortex cell types in defining the mechanical properties of hair.     

Bobcat Hair Cortisol Correlates with Land Use and Climate

Bobcats (Lynx rufus) have been increasing in abundance in the northeast United States despite a corresponding trend of increased anthropogenic land uses. Inhabiting areas of high human land use can affect stress levels, and hence cortisol titers, for wildlife species by increasing frequency of human interaction and altering habitats. In turn, increased cortisol levels can have negative effects at the individual and population level including decreased immune function, slowed growth and tissue repair, reduced reproductive capacity, and nutritional deficiencies. We quantified cortisol in bobcats across New Hampshire and Vermont, USA, using hair samples, then explored associations between hair cortisol and various organismal, land use, land cover, and climatic variables at 2 different spatial scales. Hair cortisol differed by season and bobcat mass. On average, cortisol levels were higher in fall than in spring, and larger bobcats had lower cortisol levels. Anthropogenic land uses—especially residential and agricultural uses—were the most important predictors of hair cortisol at the town scale ( area = 93 km²). At a larger scale (Wildlife Management Units; area = 1,256 km²), temperature and precipitation were better predictors of hair cortisol, suggesting that extreme weather may have significant effects on bobcat population dynamics. Our results highlight the importance of landscape composition and local conditions in the sustainable management of furbearer populations. © 2021 The Wildlife Society.