Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

High-pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle.

Cryofixation by high-pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of <200 µm thickness and about 2000 µm across. A wool follicle is a rare intact organ in a single sample about 200 µm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes occur during wool fibre development. Limited water movement and accessibility of fixatives are some issues that negatively affect the preservation of the follicle ultrastructure via conventional chemical processing. Here, we show that HPF-FS of wool follicles can yield high-quality tissue preservation for ultrastructural studies using transmission electron microscopy.

Grafting of chitosan-acrylamide hybrid on the wool: Characterization, reactive dyeing, antioxidant and antibacterial studies.

This study presents a new approach to enhance reactive dye uptake and functional finishing of wool yarns via simple grafting with synthesized chitosan-acrylamide (Ch-Ac) hybrid. To this, Ch-Ac was synthesized and characterized with fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) techniques. Then, Ch-Ac was grafted on wool, characterized with FTIR, SEM, and weight gain analysis and dyeability with two commercial reactive dyes. Results showed that Ch-Ac treated wool could be dyed at lower temperatures (ca. 40 °C), times (ca. 30 min), and amount of reactive dye (2% owf) as compared to raw wool. Also, deeper shades not obtainable in conventional dyeing could be attained using Ch-Ac treated wool. In addition, Ch-Ac treatment imparted very good radical scavenging and excellent antibacterial activity against gram-negative (E. coli) and gram-positive (S. aureus) bacteria. Color fastness results confirmed that Ch-Ac treatment had no adverse effect on durability of dyes against washing, light, rubbing and perspiration. The results of this study clearly indicated that Ch-Ac can be used in eco-friendly functional finishing of wool with enhanced reactive dye uptake, minimized residual dye in wastewater, saving in consumption of chemicals, energy, and time of dyeing.

Differences between ultrastructure and protein composition in straight hair fibres.

Mammalian hairs are internally patterned from both a morphological and proteomic perspective to exhibit specific functional traits, including curvature, which is important for coat structure affecting thermo-insulation. Most functional traits in mammalian coats are complex emergent phenomena associated with single-fibre properties that are themselves multi-variate and poorly understood. Here we compare hair curvature, ultrastructure, microstructure, protein composition and felting (a functional attribute) between fibres from natural straight-wool mutants of domestic sheep (felting lustre-mutant sheep), their wild-type relatives and also with a straight-haired semi-lustrous breed, English Leicester. Proteomic and structural results confirmed that the straight lustre mutant fibres had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type fibres, but differed from equivalent fibres from wild-type relatives and English Leicester in layout and relative proportions. While curved wild-type fibres had bilaterally arranged orthocortex and paracortex, and English Leicester fibres had a scatter of paracortex on a background of orthocortex, lustre mutant fibres typically had a complete or partial ring of orthocortex surrounding a paracortex core, and sometimes a central orthocortex (similar to straight human and goat hairs). Lustre mutant fibres also had a reduced abundance of some high glycine-tyrosine proteins, normally associated with the orthocortex, with a possible relationship between the protein expression of the KAP8 and KAP16 protein families and fibre felting properties. We conclude that through control of the internal fibre patterning, multiple-solutions to hair curvature are possible, and variation may affect mechanical phenotype differently. Felting lustre mutant sheep will be a useful tool for discriminating cause and effect from non-causative correlation in mammalian fibre development.

Helical twist direction in the macrofibrils of keratin fibres is left handed.

Macrofibrils, the main structural features within the cortical cells of mammalian hair shafts, are long composite bundles of keratin intermediate filaments (KIFs) embedded in a matrix of keratin-associated proteins. The KIFs can be helically arranged around the macrofibril central axis, making a cylinder within which KIF helical angle relative to macrofibril axis increases approximately linearly from macrofibril centre to edge. Mesophase-based self-assembly has been implicated in the early formation of macrofibrils, which first appear as liquid-crystal tactoids in the bulb of hair follicles. Formation appears to be driven initially by interactions between pre-keratinized KIFs. Differences in the nature of these KIF-KIF interactions could result in all macrofibrils being internally twisted in a single handedness, or a 50:50 mixture of handedness within each cortical cell. We data-mined 41 electron tomograms containing three-dimensional macrofibril data from previously published studies of hair and wool. In all 644 macrofibrils examined we found that within each tomogram all macrofibrils had the same handedness. We concluded that earlier reports of left- and right-handed macrofibrils were due to artefacts of imaging or data processing. A handedness marker was used to confirm (using re-imaged sections from earlier studies) that, in both human and sheep, all macrofibrils are left-handed around the macrofibril axis. We conclude that this state is universal within mammalian hair. This also supports the conclusion that the origin of macrofibril twist is the expression of chiral twisting forces between adjacent KIFs, rather than mesophase splay and bending forces relaxing to twisting forces acting within a confined space.

Fibre Ultrastructure.

Mammalian hair fibres can be structurally divided into three main components: a cuticle, cortex and sometimes a medulla. The cuticle consists of a thin layer of overlapping cells on the surface of the fibre, constituting around 10% of the total fibre weight. The cortex makes up the remaining 86-90% and is made up of axially aligned spindle-shaped cells of which three major types have been recognised in wool: ortho, meso and para. Cortical cells are packed full of macrofibril bundles, which are a composite of aligned intermediate filaments embedded in an amorphous matrix. The spacing and three-dimensional arrangement of the intermediate filaments vary with cell type. The medulla consists of a continuous or discontinuous column of horizontal spaces in the centre of the cortex that becomes more prevalent as the fibre diameter increases.

Crosslinking Between Trichocyte Keratins and Keratin Associated Proteins.

Trichocyte keratins differ considerably from their epithelial cousins in having a higher number of cysteine residues, of which the greater proportion are located in the head and tail regions of these proteins. Coupled with this is the presence of a large number of keratin associated proteins in these fibres that are high in their cysteine content, the high sulfur proteins and ultra-high sulfur proteins. Thus it is the crosslinking that occurs between the cysteines in the keratins and KAPs that is an important determinant in the functionality of wool and hair fibres. Studies have shown the majority of the cysteine residues are involved in internal crosslinking in the KAPs leaving only a few specific cysteines to interact with the keratins, with most evidence pointing to interactions between these KAP cysteines and the keratin head groups.

Comparative Study of Ultrasonication-Induced and Naturally Self-Assembled Silk Fibroin-Wool Keratin Hydrogel Biomaterials.

This study reports the formation of biocompatible hydrogels using protein polymers from natural silk cocoon fibroins and sheep wool keratins. Silk fibroin protein contains ß-sheet secondary structures, allowing for the formation of physical cross-linkers in the hydrogels. Comparative studies were performed on two groups of samples. In the first group, ultrasonication was used to induce a quick gelation of a protein aqueous solution, enhancing the ability of Bombyx mori silk fibroin chains to quickly entrap the wool keratin protein molecules homogenously. In the second group, silk/keratin mixtures were left at room temperature for days, resulting in naturally-assembled gelled solutions. It was found that silk/wool blended solutions can form hydrogels at different mixing ratios, with perfectly interconnected gel structure when the wool content was less than 30 weight percent (wt %) for the first group (ultrasonication), and 10 wt % for the second group (natural gel). Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) were used to confirm that the fibroin/keratin hydrogel system was well-blended without phase separation. Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structures of blended protein gels. It was found that intermolecular ß-sheet contents significantly increase as the system contains more silk for both groups of samples, resulting in stable crystalline cross-linkers in the blended hydrogel structures. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to analyze the samples' characteristic morphology on both micro- and nanoscales, which showed that ultrasonic waves can significantly enhance the cross-linker formation and avoid phase separation between silk and keratin molecules in the blended systems. With the ability to form cross-linkages non-chemically, these silk/wool hydrogels may be economically useful for various biomedical applications, thanks to the good biocompatibility of protein molecules and the various characteristics of hydrogel systems.

Hair microstructure of the first time found calf of woolly rhinoceros Coelodonta antiquitatis.

Hair microstructure of the first calf of the woolly rhinoceros Coelodonta antiquitatis found in Sakha in 2014 (the neck and hind leg hair) was examined by the light and electron scanning microscopy. The calf hair features were compared with those of two adults studied earlier. The calf coat color was much lighter than in adults, from pale ashy to blond. The extent of hair differentiation, dimensional and pigmentation indices were lower in the calf than in adult rhinoceroses. There was no medulla in the calf hairs, while in those of adults it was occasionally found. The cortical and cuticular layer microstructure was similar in all the animals compared. In both calf and adult hairs, there were traces of mechanical damage.

Assessing the impact of synchrotron X-ray irradiation on proteinaceous specimens at macro and molecular levels.

Synchrotron radiation (SR) has become a preferred technique for the analysis of a wide range of archeological samples, artwork, and museum specimens. While SR is called a nondestructive technique, its effect on proteinaceous specimens has not been fully investigated at the molecular level. To investigate the molecular level effects of synchrotron X-ray on proteinaceous specimens, we propose a methodology where four variables are considered: (1) type of specimen: samples ranging from amino acids to proteinaceous objects such as silk, wool, parchment, and rabbit skin glue were irradiated; (2) synchrotron X-ray energy; (3) beam intensity; (4) irradiation time. Irradiated specimens were examined for both macroscopic and molecular effects. At macroscopic levels, color change, brittleness, and solubility enhancement were observed for several samples within 100 s of irradiation. At molecular levels, the method allowed one to quantify significant amino acid modifications. Aspartic acid (Asp), wool, parchment, and rabbit skin glue showed a significant increase in Asp racemization upon increasing irradiation time with rabbit skin glue showing the greatest increase in d-Asp formation. In contrast, Asp in silk, pure cystine (dimer of cysteine), and asparagine (Asn) did not show signs of racemization at the irradiation times studied; however, the latter two compounds showed significant signs of decomposition. Parchment and rabbit skin glue exhibited racemization of Asp, as well as racemization of isoleucine (Ile) and phenylalanine (Phe) after 100 s of irradiation with a focused beam. Under the experimental conditions and sample type and dimensions used here, more change was observed for focused and low energy (8 keV) beams than unfocused or higher energy (22 keV) beams. These results allow quantification of the change induced at the molecular level on proteinaceous specimens by synchrotron X-ray radiation and help to define accurate thresholds to minimize the probability of damage occurring to cultural heritage specimens. For most samples, damage was usually observed in the 1-10 s time scale, which is about an order of magnitude longer than SR studies of cultural heritage under X-ray fluorescence (XRF) mode; however, it is consistent with the duration of X-ray absorption spectroscopy (XAS) and microcomputed tomography (µCT) measurements.

Field-emission scanning electron microscopy and energy-dispersive x-ray analysis to understand the role of tannin-based dyes in the degradation of historical wool textiles.

An innovative approach, combining field-emission scanning electron microscopy (FESEM) with energy dispersive X-ray spectroscopy (EDX) analysis, is presented to investigate the degradation mechanisms affecting tannin-dyed wool. In fact, tannin-dyed textiles are more sensitive to degradation then those dyed with other dyestuffs, even in the same conservation conditions. FESEM-EDX was first used to study a set of 48 wool specimens (artificially aged) dyed with several raw materials and mordants, and prepared according to historical dyeing recipes. EDX analysis was performed on the surface of wool threads and on their cross-sections. In addition, in order to validate the model formulated by the analysis of reference materials, several samples collected from historical and archaeological textiles were subjected to FESEM-EDX analysis. FESEM-EDX investigations enabled us to reveal the correlation between elemental composition and morphological changes. In addition, aging processes were clarified by studying changes in the elemental composition of wool from the protective cuticle to the fiber core in cross-sections. Morphological and elemental analysis of wool specimens and of archaeological and historical textiles showed that the presence of tannins increases wool damage, primarily by causing a sulfur decrease and fiber oxidation.

Internal lipids of felted, yellowed and pathologically thin wool.

The keratin fibers contain small amount of the internal lipids which are in free state or bound with fiber proteins via tioester of 18-methyleicosanoic acid. Today the origin of these lipids, their composition and functional properties are still not found. Therefore, our objective was to examine the content and composition of internal lipids in sheep's wool with different defects. We observed that regardless of the type of fibers defect there are significant changes especially in the quality composition of the internal lipids, although the total content of free and covalently bound lipids in all cases is practically identical. Notably, both free and covalently bound lipids composition of felted and simultaneously felted and yellowed wool is characterized by changes in contents mainly of free fatty acids and ceramides whereas abnormal thinning of fibers is accompanied only by a decrease of sulfolipids.

A novel method for identifying shahtoosh.

Shahtoosh, the down hair of the Tibetan antelope (Pantholops hodgsonii), is the noblest and most expensive wool in the world. The population of the animal has declined dramatically due to commercial poaching for the fiber. Traditional inspection for detection of shahtoosh has been performed by microscopic analysis. We developed a TaqMan real-time PCR-based DNA analysis method for identifying shahtoosh fibers. A set of probe and primers for the mitochondrial 12S ribosomal RNA gene that binds specifically to Tibetan antelope DNA was designed. A signal was detected with sensitivity to the 1:10,000 dilution of shahtoosh DNA. A fiber mixture of 1% of shahtoosh mixed with cashmere and even a single fiber can be detected with this method. The method is faster, more cost-effective and more sensitive than other traditional sequencing methods and can be directly applied to identify shahtoosh and its processed products, which will be of value in illegal trade investigations.

[Cellular dynamics of the outer layers of the hair follicle of fine-wool sheep during the phase of stable hair growth].

The structure, origin, and migration of outer sheath cells of the hair follicles of domestic sheep were studied by electron microscopic, autoradiographic, and histochemical (glycogen) in order to understand the role of this layer in hair morphogenesis. We demonstrated that the cells of the outer layers of the outer sheath interpose into the inner "companion" layer of the outer sheath. Although this process takes place all along the hair follicle from the lower bulb up to the sebaceous glands orifices, it mainly takes place over the bulb. Labeled cells interposed into the companion layer move towards sebaceous glands orifices more than 24 hours faster than labeled cells of the inner sheath and hair, because these cells included the label not in the bulb cambium (as hair and inner sheath) but over the bulb, and from this point they start movement. Interposition of cells into the companion layer must cause increase of its volume and additional volume supposed to be led away into the pillar canal around the hair near the sebaceous glands orifices. This can provide the mechanism for the propagation of the hair and inner sheath promotion to sebaceous gland orifices.

Sequence analysis and heterologous expression of the wool cuticle-degrading enzyme encoding genes in Fusarium oxysporum 26-1.

Two protease-like proteins, KrtA and KrtC, were identified in Fusarium oxysporum 26-1. Genes coding these proteins, krtA and krtC, were isolated and characterized. Recombinant KrtA (rKrtA) and KrtC (rKrtC) were successfully expressed in Aspergillus oryzae and secreted. The combination of rKrtA and rKrtC completely removed the cuticle of wool fibers.

Antimicrobial wool, polyester and a wool/polyester blend created by silver particles embedded in a silica matrix.

A two-step antimicrobial finishing procedure was applied to wool (WO) and polyester (PES) fabrics and a WO/PES fabric blend, in which the pad-dry-cure method was performed to create a functional silica matrix through the application of an inorganic-organic hybrid sol-gel precursor (RB) followed by the in situ synthesis of AgCl particles on the RB-treated fibres using 0.10 and 0.50mM AgNO3 and NaCl. The bulk concentration of Ag on the cotton fibres was determined by inductively coupled plasma mass spectroscopy. The antimicrobial activity was determined for the bacteria Escherichia coli and Staphylococcus aureus, and the fungus Aspergillus niger. The results showed that the highest concentration of the adsorbed Ag compound particles was on the WO samples followed by the WO/PES and PES samples. The antimicrobial activity of the finished fabric samples strongly depended not only on the amount of adsorbed Ag but also on the properties of the fabric samples. Whereas Ag biocidal activity was generated for the finished PES samples at Ag particle concentrations of less than 10mg/kg, the 34-times higher Ag particle concentration on the WO samples was insufficient to impart satisfactory antimicrobial activity because Ag chemically binds to the thiol groups on wool. The presence of wool fibres in WO/PES samples decreased the antimicrobial protection of the fabric blend compared with that of the PES fabric. A lethal concentration of adsorbed Ag compound particles for bacteria and fungi was produced only through the treatment of the WO and WO/PES samples with 0.5mM AgNO3.

Layer-by-layer self-assembly immobilization of catalases on wool fabrics.

A new immobilization strategy of catalases on natural fibers was reported in this paper. Catalase (CAT) from Bacillus subtilis was assembled into multiple layers together with poly(diallyldimethylammonium chloride) (PDDA) on wool fabrics via layer-by-layer (LBL) electrostatic self-assembly deposition. The mechanism and structural evaluation of LBL electrostatic self-assembly were studied in terms of scanning electron microscopy (SEM), surface zeta potential, and apparent color depth (K/S). The SEM pictures showed obvious deposits absorbed on the wool surfaces after LBL self-assembly. The surface zeta potential and dyeing depth of CAT/PDDA-assembled wool fabrics presented a regular layer-by-layer alternating trend along with the change of deposited materials, revealing the multilayer structure of the wool fiber immobilized catalases. The V(max) values were found to be 2,500±238 U/mg protein for the free catalase and 1,000±102 U/mg protein for the immobilized catalase. The K(m) value of free catalase (11.25±2.3 mM) was found to be lower than that of the immobilized catalase (222.2±36.5 mM). The immobilized catalase remained high enzymatic activity and showed a measureable amount of reusability, which proved that LBL electrostatic self-assembly deposition is a promising approach to immobilize catalases.

Interspecies comparison of morphology, ultrastructure, and proteome of mammalian keratin fibers of similar diameter.

Sheep wool has traditionally been viewed as the representative mammalian keratin fiber for the purposes of describing morphology and protein composition. We have investigated narrow fibers from the under-hairs of a range of species both closely and distantly related to sheep, comparing structure and protein composition. Within this group, curvature was negatively correlated with diameter for all but mohair. The cortical cell types present in alpaca, rabbit, and mohair fibers differed structurally from wool, primarily in terms of their macrofibril architecture. Except for rabbit, each species' fibers contained three cell types, and except for mohair, cell types were distributed asymmetrically across the cortex. In mohair, the cell types were distributed annularly, and each cell type had regions in which intermediate filaments were packed into highly aligned hexagonal mosaics, much like the mesocortex in wool. Coupled with this, were differences in the protein profiles; the rabbit fiber contained extra keratins and keratin associated proteins, while only subtle differences were noted between mohair and Merino fibers. In both rabbit and mohair fibers, the relative abundance of keratin K85 was lower than that of Merino. These results suggest that there may be links between relative protein composition and fiber morphology, albeit complex ones.

Molecular structure and properties of wool fiber surface-grafted with nano-antibacterial materials.

Wool fiber was modified by ultraviolet irradiation (UV) and functionalized by grafting antibacterial agent. The structure and properties of antibacterial wool fiber were discussed in detail. The secondary structure changes and crystal structure were analyzed based on Fourier Transformation Raman Spectrometry (FTR) and X-ray diffraction (XRD). The results show that the disordered degree of UV-treated sample was increased and the antibacterial sample became more oriented. Compared with parent wool fiber, the antibacterial wool fiber was improved in mechanical property. The force, tensile strength and elongation were increased by 18%, 16%, and 7%, respectively. Also, the anti-shrinkage performance was increased because of the decrease in the directional frictional effect (DFE).