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Keratins, the most abundant proteins in human hair, are excellent hair nutrients for growth. However, the complex components of keratin extract hinder their mechanism investigation, and the pure recombinant keratin with poor solubility limited its hair growth promotion efficiency. Here, the water-soluble recombinant keratins (RKs) of K31 and K81 are rationally designed through QTY Code methodology, which are then used to fabricate the microneedles to study the effect of keratin on hair growth. Interestingly, it is discovered that more than 40% of the hair follicles (HFs) in the RK81 QTY group entered the anagen on day 12 and the diameter of new hair is 15.10 ± 2.45 µm, which significantly promoted growth and development of HFs and improved new hair quality compared to RK31 QTY . Water-soluble RKs significantly enhanced HFs activity and de novo regeneration of robust hairs compared to extract and minoxidil by upregulating the PI3K/AKT/Nf-κB signaling axis. These findings highlight the potential of designing solubilized recombinant keratins with distinct properties to improve therapeutical effects and open new avenues to designing keratin-based proteins., (© 2024 Wiley‐VCH GmbH.)

Human hair keratin (HHK) has been successfully explored as raw materials for three-dimensional scaffolds for soft tissue regeneration due to its excellent biocompatibility and bioactivity. However, none of the reported HHK based scaffolds is able to replicate the strain-stiffening capacity of living tissues when responding to large deformations. In the present study, strain-stiffening property was achieved in scaffolds fabricated from HHK via a synergistic effect of well-defined, aligned microstructure and chemical crosslinking. Directed ice-templating method was used to fabricate HHK-based scaffolds with highly aligned (anisotropic) microstructure while oxidized dopamine (ODA) was used to crosslink covalently to HHKs. The resultant HHK-ODA scaffolds exhibited strain-stiffening behavior characterized by the increased gradient of the stress-strain curve after the yield point. Both ultimate tensile strength and the elongation at break were enhanced significantly (~700 kPa, ~170%) in comparison to that of HHK scaffolds lacking of aligned microstructure or ODA crosslinking. In vitro cell culture studies indicated that HHK-ODA scaffolds successfully supported human dermal fibroblasts (HDFs) adhesion, spreading and proliferation. Moreover, anisotropic HHK-ODA scaffolds guided cell growth in alignment with the defined microstructure as shown by the highly organized cytoskeletal networks and nuclei distribution. The findings suggest that HHK-ODA scaffolds, with strain-stiffening properties, biocompatibility and bioactivity, have the potential to be applied as biomimetic matrices for soft tissue regeneration., (© 2021 Wiley Periodicals LLC.)

Traumatic injury of the oral cavity is atypical and often accompanied by uncontrolled bleeding and inflammation. Injectable hydrogels have been considered to be promising candidates for the treatment of oral injuries because of their simple formulation, minimally invasive application technique, and site-specific delivery. Fibrinogen-based hydrogels have been widely explored as effective materials for wound healing in tissue engineering due to their uniqueness. Recently, an injectable foam has taken the spotlight. However, the fibrin component of this biomaterial is relatively stiff. To address these challenges, we created keratin-conjugated fibrinogen (KRT-FIB). This study aimed to develop a novel keratin biomaterial and assess cell-biomaterial interactions. Consequently, a novel injectable KRT-FIB hydrogel was optimized through rheological measurements, and its injection performance, swelling behavior, and surface morphology were investigated. We observed an excellent cell viability, proliferation, and migration/cell-cell interaction, indicating that the novel KRT-FIB-injectable hydrogel is a promising platform for oral tissue regeneration with a high clinical applicability.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament., (© 2021 Wiley Periodicals LLC.)

The main core of wound treatment is cell growth and anti-infection. To accelerate the proliferation of fibroblasts in the wound and prevent wound infections, various strategies have been tried. It remains a challenge to obtain good cell proliferation and antibacterial effects. Here, human hair kerateine (HHK)/poly(ethylene oxide) (PEO)/poly(vinyl alcohol) (PVA) nanofibers were prepared using cysteine-rich HHK, and then, silver nanoparticles (AgNPs) were in situ anchored in the sulfur-containing amino acid residues of HHK. After the ultrasonic degradation test, HHK/PEO/PVA nanofibrous mats treated with 0.005-M silver nitrate were selected due to their relatively complete structures. It was observed by TEM-EDS that the sulfur-containing amino acids in HHK were the main anchor points of AgNPs. The results of FTIR, XRD and the thermal analysis suggested that the hydrogen bonds between PEO and PVA were broken by HHK and, further, by AgNPs. AgNPs could act as a catalyst to promote the thermal degradation reaction of PVA, PEO and HHK, which was beneficial for silver recycling and medical waste treatment. The antibacterial properties of AgNP-HHK/PEO/PVA nanofibers were examined by the disk diffusion method, and it was observed that they had potential antibacterial capability against Gram-positive bacteria, Gram-negative bacteria and fungi. In addition, HHK in the nanofibrous mats significantly improved the cell proliferation of NIH3T3 cells. These results illustrated that the AgNP-HHK/PEO/PVA nanofibrous mats exhibited excellent antibacterial activity and the ability to promote the proliferation of fibroblasts, reaching our target applications.

Human hair keratin (HHK) is successfully exploited as raw materials for 3D scaffolds for soft tissue regeneration owing to its excellent biocompatibility and bioactivity. However, most HHK scaffolds are not able to achieve the anisotropic mechanical properties of soft tissues such as tendons and ligaments due to lack of tunable, well-defined microstructures. In this study, directed ice templating method is used to fabricate anisotropic HHK scaffolds that are characterized by aligned pores (channels) in between keratin layers in the longitudinal plane. In contrast, pores in the transverse plane maintain a homogenous rounded morphology. Channel widths throughout the scaffolds range from ≈5 to ≈15 µm and are tunable by varying the freezing temperature. In comparison with HHK scaffolds with random, isotropic pore structures, the tensile strength of anisotropic HHK scaffolds is enhanced significantly by up to fourfolds (≈200 to ≈800 kPa) when the tensile load is applied in the direction parallel to the aligned pores. In vitro results demonstrate that the anisotropic HHK scaffolds are able to support human dermal fibroblast adhesion, spreading, and proliferation. The findings suggest that HHK scaffolds with well-defined, aligned microstructure hold promise as templates for soft tissues regeneration by mimicking their anisotropic properties., (© 2020 Wiley-VCH GmbH.)

Many restoring formulations for damaged hair keratin have been developed. Some patents claim that the hair repair occurs through the reconstruction of disulfide bridges of keratin, through α,β-unsaturated Michael acceptors, such as shikimic acid and bis-aminopropyl diglycol dimaleate. To gain more insights into the possible repairing mechanism, this study is aimed at assessing, by IR and Raman spectroscopies coupled to scanning electron microscopy (SEM), the structural changes induced in keratin from bleached hair by the treatment with commercial reconstructive agents as well as shikimic acid and dimethyl maleate, chosen as model compounds. Vibrational spectroscopy revealed that shikimic acid- and maleate-based restoring agents interacted with hair fibers modifying both their cortex and cuticle regions. None of the investigated treatments induced an increase in the SS disulfide bridges content of the hair cortex, although it cannot be excluded that this phenomenon could have occurred in the cuticle. SS rearrangements were found to occur. None of our results can be interpreted as direct evidence of the sulfa-Michael reaction/cross-linking. From a morphological point of view, beneficial effects of the restoring agents were observed by SEM analyses, in terms of a more regular hair surface and more imbricated scales., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Natural biopolymers have found success in tissue engineering and regenerative medicine applications. Their intrinsic biocompatibility and biological activity make them well suited for biomaterials development. Specifically, keratin-based biomaterials have demonstrated utility in regenerative medicine applications including bone regeneration, wound healing, and nerve regeneration. However, studies of structure-function relationships in keratin biomaterials have been hindered by the lack of homogeneous preparations of materials extracted and isolated from natural sources such as wool and hair fibers. Here we present a side-by-side comparison of natural and recombinant human hair keratin proteins K31 and K81. When combined, the recombinant proteins (i.e. rhK31 and rhK81) assemble into characteristic intermediate filament-like fibers. Coatings made from natural and recombinant dimers were compared side-by-side and investigated for coating characteristics and cell adhesion. In comparison to control substrates, the recombinant keratin materials show a higher propensity for inducing involucrin and hence, maturation in terms of potential skin cell differentiation.

Keratins derived from human hair possess excellent wound healing qualities. However, their functional contribution to this process is poorly understood. In this study, the regulatory function of human hair keratins in wound healing is investigated using proteomic analysis by dividing keratins into different groups based on their molecular weight distributions: low molecular weight keratins (LMWK, 10-30 kDa), medium molecular weight keratins (MMWK, 30-50 kDa), and high molecular weight keratins (HMWK, >50 kDa). Keratin hydrogels with different molecular weights exhibit various morphologies, rheological properties, degradation rates, and wound healing activities. Using proteomic analysis, LMWK and HMWK hydrogels exhibit a stronger regulatory ability for wound healing at days 1 and 7, respectively. The major functions of LMWK during wound healing are regulation of cells communication and function. In contrast, proteins associated with energy metabolism are significantly expressed after HMWK hydrogel treatment at day 1, and these play an important role in cellular growth and reactive oxygen species scavenging at day 7. These results demonstrate that the wound healing qualities of human hair keratins are influenced by their molecular weight distribution, and the proteomic analysis sheds new light on the regulatory function of human hair keratins during wound healing., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study offers a new antibacterial wound dressing from carboxymethyl cellulose (CMC)-human hair keratin with topical clindamycin delivery. Keratin was successfully extracted from human hair. Different sponges fabricated by changing CMC to keratin ratio were characterized and compared. Halloysite nanotubes were used as carriers to control the clindamycin release. Various characterization techniques were used to determine the effects of keratin addition on the structure, morphology, physical properties, drug release, antibacterial activity, and cellular behavior of CMC hydrogels. As proved by SEM and EDS, porous structure with interconnected pores was successfully formed and clindamycin-loaded HNTs were uniformly dispersed within the porous structures. Increasing the keratin in CMC hydrogel not only lowered its water vapor transmission rate to a suitable range for wound healing but also improved the water stability of CMC hydrogel. The in vitro release study indicated that clindamycin was released slower in samples containing higher keratin and the Fickian diffusion mechanism controlled their release profile. The fabricated dressing effectively inhibits S. aureus bacterial colonies growth after 24 h. Fibroblast culturing on the fabricated sponges indicated that cellular attachment, proliferation, and spreading were significantly enhanced with increasing the keratin amount., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Keratins derived from human hair have been suggested to be particularly effective in general surgical wound healing. However, the healing of a combined radiation-wound injury is a multifaceted regenerative process. Here, hydrogels fabricated with human hair keratins were used to test the wound healing effects on rats suffering from combined radiation-wound injuries. Briefly, the keratin extracts were verified by dodecyl sulfate polyacrylamide gel electrophoresis analysis and amino acid analysis, and the keratin hydrogels were then characterized by morphological observation, Fourier transform infrared spectroscopy analysis and rheology analyses. The results of the cell viability assay indicated that the keratin hydrogels could enhance cell growth after radiation exposure. Furthermore, keratin hydrogels could accelerate wound repair and improve the survival rate in vivo. The results demonstrate that keratin hydrogels possess a strong ability to accelerate the repair of a combined radiation-wound injury, which opens up new tissue regeneration applications for keratins.

The keratin-based scaffolds are getting more and more attention in the application of tissue engineering. Though various approaches have been considered to improve the physical properties of these scaffolds, few succeeded in achieving the enhanced properties of the pure keratin scaffolds. Due to the presence of -OH, -NH 2 , >CO, and -SH on the extracted human hair keratin (HHK), the formation of hydrogen bonds and disulfide bridges could be triggered under certain conditions, leading to the self-cross-linking of HHK materials. Herein, a simple and green strategy was introduced, via freeze-thaw cycles of keratin solutions without addition of extraneous reagents, to obtain the mechanically robust HHK scaffolds. The comparative quantitation of residual -SH among the samples treated with 1, 5, and 9 cycles confirmed the oxidation in the thaw process for forming disulfide bonds. So, the equivalent thaw time was applied in this study, and three groups of the treated samples after 1, 5, and 9 cycles with an appropriate extension thaw time were prepared to solely investigate the effects of physical cross-linking networks, primarily by formation of hydrogen bonds, on the properties of the obtained scaffolds. The systematic assessments including swelling behavior, porosity, thermal analysis, compressive measurement, and microstructural observation confirmed that the repetitive freeze-thaw treatment contributed to mechanically robust scaffolds with good porous interconnectivity. The cell culturing experiments further verified that these HHK scaffolds had desirable cytocompatibility, permitting the proper proliferation, attachment, and infiltration. Accordingly, this study provided a simple and efficient method to obtain biocompatible, mechanically robust keratin scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1452-1461, 2019., (© 2018 Wiley Periodicals, Inc.)

In recent years, favorable enhanced wound-healing properties and excellent biocompatibility of keratin derived from human hair have attracted considerable attention. Recombinant keratin proteins can be produced by recombinant DNA technology and have higher purity than extracted keratin. However, the wound-healing properties of recombinant keratin proteins remain unclear. Herein, two recombinant trichocyte keratins including human type I hair keratin 37 and human type II hair keratin 81 were expressed using a bacterial expression system, and recombinant keratin nanoparticles (RKNPs) were prepared via an ultrasonic dispersion method. The molecular weight, purity, and physicochemical properties of the recombinant keratin proteins and nanoparticles were assessed using gel electrophoresis, circular dichroism, mass spectrometry, and scanning electron microscope analyses. The RKNPs significantly enhanced cell proliferation and migration in vitro, and the treatment of dermal wounds in vivo with RKNPs resulted in improved wound healing associated with improved epithelialization, vascularization, and collagen deposition and remodeling. In addition, the in vivo biocompatibility test revealed no systemic toxicity. Overall, this work demonstrates that RKNPs are a promising candidate for enhanced wound healing, and this study opens up new prospects for the development of keratin biomaterials.

The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections z a  ∼ 74 Å and z b  ∼ 198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The intrinsically high cysteine content in human hair keratins and keratin associated proteins confer hair its outstanding mechanical strength through the formation of strong intermolecular disulfide bonds. In addition, these proteins offer the potential to be exploited as potent antioxidants. This report presents our findings on the antioxidant effects of human hair protein extracts and their consequent protective role against oxidative stress in human dermal fibroblast (HDF) cultures. Protein extracts were obtained from human hair using sodium sulfide as the reducing agent, and characterized using SDS-PAGE, Western blotting, MALDI-ToF mass spectrometry and amino acid analysis. Cysteine was found to account for 11.2 mol % in the extracted fractions. By measuring 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, the hair protein fractions were shown to possess significant antioxidant ability (IC 50  = 16.22 μM). As a supplement in cell culture media, the extracts protected HDFs from H 2 O 2 induced oxidative stress, which was demonstrated by the maintenance of cell viability and reduced reactive oxygen species production. Besides offering mechanical support as a scaffolding material, the unique antioxidizing ability of human hair protein extracts may also be exploited in biomedical applications.

The major components of hair are keratins and keratin associated proteins (KRTAPs). KRTAPs form the interfilamentous matrix between intermediate filament bundles through extensive disulfide bond cross-linking with the numerous cysteine residues in hair keratins. A variable number of approximately100-180 genes compose the KRTAP gene family in mammals. KRTAP gene family members present a typical pattern of concerted evolution, and its evolutionary features are consistent with the evolution of mammalian hair. KRATP genes might be more important in determining the structure of cashmere fibers in domestic mammals like sheep and goats. KRTAP gene variants thus should provide information for improved wool by sheep and goat breeding.

Wool and hair fibres are primarily composed of proteins of which the keratins and keratin associated proteins (KAPs) are the major component. Considerable diversity is known to exist within these two groups of proteins. In the case of the keratins two major families are known, of which there are 11 members in the acidic Type I family and 7 members in the neutral-basic Type II family. The KAPs are even more diverse than the keratins, with 35 families being known to exist when the KAPs found in monotremes, marsupials and other mammalian species are taken into consideration. Human hair and wool are known to have 88 and 73 KAPs respectively, though this number rises for wool when polymorphism within KAP families is included.

Biomaterials have been attracting attention as a useful building block for biocompatible and bioresorbable electronics due to their nontoxic property and solution processability. In this work, we report the integration of biocompatible keratin from human hair as dielectric layer for organic thin-film transistors (TFTs), with high performance, flexibility, and transient property. The keratin dielectric layer exhibited a high capacitance value of above 1.27 μF/cm 2 at 20 Hz due to the formation of electrical double layer. Fully solution-processable TFTs based on p-channel poly[4-(4,4-dihexadecyl-4H-cyclopenta[1,2-b:5,4-b]dithiophen-2-yl)-alt[1,2,5]thiadiazolo[3,4-c]-pyridine] (PCDTPT) and keratin dielectric exhibited high electrical property with a saturation field-effect mobility of 0.35 cm 2 /(Vs) at a low gate bias of -2 V. We also successfully demonstrate flexible TFTs, which exhibited good mechanical flexibility and electrical stability under bending strain. An artificial electronic synaptic PCDTPT/keratin transistor was also realized and exhibited high-performance synaptic memory effects via simple operation of proton conduction in keratin. An added functionality of using keratin as a substrate was also presented, where similar PCDTPT TFTs with keratin dielectric were built on top of keratin substrate. Finally, we observed that our prepared devices can be degraded in ammonium hydroxide solution, establishing the feasibility of keratin layer as various components of transient electrical devices, including as a substrate and dielectric layer.

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by-products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero- and one-dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self-assembly mechanism highlights the importance of disulfide crosslinking in keratin self-assembly., (© 2017 Wiley Periodicals, Inc.)

For the past 50years there has been considerable debate over the sub-structure of the fully differentiated (oxidised) trichocyte keratin intermediate filament. Depending on the staining and preparative procedures employed, IF observed in transverse section in the transmission electron microscope have varied in appearance between that of a "ring" and a "ring-core" structure, corresponding to the so-called (8+0) and (7+1) protofilament arrangements. In a new analysis of the fine structure of the 1nm equatorial region of the X-ray diffraction pattern of quill we show that the observed pattern is consistent with the (8+0) model and we are also able to assign values to the various parameters. In contrast, we show that the observed X-ray pattern is inconsistent with a (7+1) arrangement. Furthermore, in the (7+1) model steric hindrance would be encountered between the core protofilament and those constituting the ring. The appearance of a central "core" in transverse TEM sections, previously attributed to a central protofilament, is explained in terms of portions of the apolar, disulfide-bonded head and/or tail domains of the trichocyte keratin IF molecules, including the conserved H subdomains, lying along the axis of the IF, thereby decreasing the efficacy of the reducing agents used prior to staining. The H1 subdomain, previously shown to be important in the assembly of epidermal IF molecules at the two- to four-molecule level, is likely to have a similar role for the trichocyte keratins and may form part of a central scaffold on which the molecules assemble into fully functional IF., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We selected 1235 decapeptides from human hair proteins encoded by human genes of keratins and keratin associated proteins. The peptides were linked to glass arrays and screened for their affinity towards a solution of human hair extracted keratin fraction. Based on the physicochemical properties of the peptides, ten variables were studied: content of different types of amino acid side chains (cysteine, hydrophobic, polar, basic, acidic, aromatic rings, amide, alcohol side chains), isoelectric point, and net charge. We found differences statistically significant on the binding affinity of peptides based on their content of cysteine, hydrophobic and polar amino acids, mainly containing alcohols. These results point to the formation of hydrophobic interactions and disulfide bonds between small peptides and human hair keratins as the main driving forces for the interaction of possible cosmetic peptides, namely designed to strength human hair. As so, our results enlighten the nature of the interaction of keratin based materials with human hair, which are claimed to enhance hair fiber strength, and enable a more directed and sustained hair care peptide design., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study, we aimed at fabricating an osteoinductive biocomposite scaffold using keratin obtained from human hair, jellyfish collagen and eggshell-derived nano-sized spherical hydroxyapatite (nHA) for bone tissue engineering applications. Keratin, collagen and nHA were characterized with the modified Lowry method, free-sulfhydryl groups and hydroxyproline content analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and thermal gravimetric analysis (TGA) which confirmed the success of the extraction and/or isolation processes. Human adipose mesenchymal stem cells (hAMSCs) were isolated and the cell surface markers were characterized via flow cytometry analysis in addition to multilineage differentiation capacity. The undifferentiated hAMSCs were highly positive for CD29, CD44, CD73, CD90 and CD105, but were not seen to express hematopoietic cell surface markers such as CD14, CD34 and CD45. The cells were successfully directed towards osteogenic, chondrogenic and adipogenic lineages in vitro. The microarchitecture of the scaffolds and cell attachment were evaluated using scanning electron microscopy (SEM). The cell viability on the scaffolds was assessed by the MTT assay which revealed no evidence of cytotoxicity. The osteogenic differentiation of hAMSCs on the scaffolds was determined histologically using alizarin red S, osteopontin and osteonectin stainings. Early osteogenic differentiation markers of hAMSCs were significantly expressed on the collagen-keratin-nHA scaffolds. In conclusion, it is believed that collagen-keratin-nHA osteoinductive biocomposite scaffolds have the potential of being used in bone tissue engineering., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by (31)P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 min for cationic moiety like methylene blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function., (© 2016 Elsevier Inc. All rights reserved.)

Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or "capping") of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10(-4) M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7) M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5) M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.

Human hair keratins are readily available, easy to extract, and eco-friendly materials with natural bioactivities. Keratin-based materials have been studied for applications such as cell culture substrates, internal hemostats for liver injury, and conduits for peripheral nerve repair. However, there are limited reports of using keratin-based 3D scaffolds for cell culture in vitro. Here, we describe the development of a 3D hair keratin hydrogel, which allows for living cell encapsulation under near physiological conditions. The convenience of making the hydrogels from keratin solutions in a simple and controllable manner is demonstrated, giving rise to constructs with tunable physical properties. This keratin hydrogel is comparable to collagen hydrogels in supporting the viability and proliferation of L929 murine fibroblasts. Notably, the keratin hydrogels contract less significantly as compared to the collagen hydrogels, over a 16-day culture period. In addition, preliminary in vivo studies in immunocompetent animals show mild acute host tissue response. These results collectively demonstrate the potential of cell-loaded keratin hydrogels as 3D cell culture systems, which may be developed for clinically relevant applications.

The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α-helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained., (© 2014 Wiley Periodicals, Inc.)

Trichocyst-enriched fractions were isolated from the marine dinophyte Prorocentrum micans. Transmission electron microscopy revealed that most of the trichocysts were discharged and had elongated to long filaments. Some trichocysts were still condensed. Fragments of discharged trichocysts measured up to 20 μm in length and 260 nm in width, those still condensed measured up to 1 μm in width and 16 μm in length. A distinct banding pattern with a transversal periodicity of approximately 16-18 nm and a periodic longitudinal striation of 3-4 nm could be measured along the trichocyst filaments. At higher magnifications, a fragile, alveolated, net-like organisation became obvious which resembled the one shown for the trichocysts of ciliates. When trichocyst-enriched fractions were treated with sodium dodecyl sulfate and centrifuged subsequently, no trichocysts were registered any longer in the sodium dodecyl sulfate-insoluble fraction by electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of trichocyst-enriched fractions and of the SDS-soluble fractions revealed a protein banding pattern which was dominated by polypeptides of 50-30, 12.5, and approximately 8.5 kDa. The polypeptide banding pattern deviated significantly from those registered for ejectisomes of cryptophytes and of the prasinophyte Pyramimonas grossii, for the Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis, and also from the banding pattern of trichocysts of Paramecium. An antiserum directed against trichocysts of Paramecium did not cross-react with the polypeptides present in the trichocyst-enriched fraction of Prorocentrum micans.

The primary function of hair and fur covering mammalian skin is to provide mechanical and thermal protection for the body. The proteins that constitute hair are extremely resistant to degradation by environmental factors. However, even durable materials can be slowly broken down by mechanical stresses, biodegradation mediated by endogenous enzymes in the skin or host microbes. We hypothesised that the biodegradation products of hair may possess bioprotective properties, which supplement their physical protective properties. Although evolutionary processes have led to a reduction in the amount of hair on the human body, it is possible that the bioprotective properties of hair biodegradation products have persisted. The human skin is exposed to various environmental carcinogenic factors. Therefore, we hypothesised that the potential bioprotective mechanisms of hair degradation products affect melanoma growth. We used pepsin to partially digest hair enzymatically, and this process produced a water-soluble lysate containing a mixture of peptides, including fragments of keratin and keratin-associated proteins. We found out that the mixtures of soluble peptides obtained from human hair inhibited the proliferation of human melanoma cells in vitro. Moreover, the hair-derived peptide mixtures also inhibited the proliferation of B lymphoma cells and urinary bladder cancer cells. Normal human cells varied in their susceptibility to the effects of the lysate; the hair-derived peptide mixtures modulated the proliferation of normal human fibroblasts but did not inhibit the proliferation of human mesenchymal cells derived from umbilical cord stromal cells. These results suggest that hair-derived peptides may represent a new class of anti-proliferative factors derived from basically structural proteins. Identification of active regulatory compounds and recognition of the mechanism of their action might pave the way to elaboration of new anticancer drugs.

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.

Since their first finding in wool 50years ago, keratin-associated proteins (KAPs), which are classified into three groups; high sulfur (HS) KAPs, ultra high sulfur (UHS) KAPs, and high glycine-tyrosine (HGT) KAPs, have been the target of curiosity for scientists due to their characteristic amino acid sequences. While HS and UHS KAPs are known to function in disulfide bond crosslinking, the function of HGT KAPs remains unknown. To clarify the function as well as the binding partners of HGT KAPs, we prepared KAP8.1 and other KAP family proteins, the trichocyte intermediate filament proteins (IFP) K85 and K35, the head domain of K85, and the C subdomain of desmoplakin C-terminus (DPCT-C) and investigated the interactions between them in vitro. Western blot analysis and isothermal titration calorimetry (ITC) indicate that KAP8.1 binds to the head domain of K85, which is helically aligned around the axis of the intermediate filament (IF). From these results and transmission electron microscopy (TEM) observations of bundled filament complex in vitro, we propose that the helical arrangement of IFs found in the orthocortex, which is uniquely distributed on the convex fiber side of the hair, is regulated by KAP8.1. Structure-dependent binding of DPCT-C to trichocyte IFP was confirmed by Western blotting, ITC, and circular dichroism. Moreover, DPCT-C also binds to some HGT KAPs. It is probable that such bidirectional binding property of HGT KAPs contribute to the mechanical robustness of hair., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A novel model of infected nail plate for testing the efficacy of topical antifungal formulations has been developed. This model utilized keratin film made of human hair keratin as a nail plate model. Subsequent to infection by Trichophyton rubrum, the common causative agent of onychomycosis, keratin films as infected nail plate models were treated with selected topical formulations, that is cream, gel, and nail lacquer. Bovine hoof was compared to keratin film. In contrast to the common antifungal susceptibility test, the antifungal drugs tested were applied as ready-to-use formulations because the vehicle may modify and control the drug action both in vitro and in vivo. Extrapolating the potency of an antifungal drug from an in vitro susceptibility test only would not be representative of the in vivo situation since these drugs are applied as ready-to-use formulations, for example as a nail lacquer. Although terbinafine has been acknowledged to be the most effective antifungal agent against T. rubrum, its antifungal efficacy was improved by its incorporation into an optimal formulation. Different gels proved superior to cream. Therefore, this study is able to discriminate between efficacies of different topical antifungal formulations based on their activities against T. rubrum., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Hair and epithelial keratins constitute the major structural components of the skin and its appendages, including the hair fibre. While it is appreciated that selected steroid hormones regulate specific keratins, little is known about the neuroendocrine control of human hair keratin expression. Preliminary evidence had suggested that thyrotropin-releasing hormone (TRH) may regulate keratin gene transcription., Objectives: To clarify whether TRH operates as a novel neuroendocrine regulator of human hair and epithelial keratin expression under physiologically relevant conditions in situ., Methods: Microdissected human female scalp hair follicles (HFs) and female scalp skin were treated in serum-free organ culture for 12 h to 6 days with 100 ng mL(-1) TRH or vehicle. Both quantitative immunohistomorphometry and quantitative real-time polymerase chain reaction were utilized to assess expression of selected keratins., Results: TRH significantly increased expression of the hair keratins K31 and K32, while that of K85 and K86, and of the epithelial keratins K14 and K17, was reduced. In the interfollicular epidermis, TRH stimulated expression of K6, K14 and K17, both at the mRNA and protein levels. Stimulation of the same keratins was also evident in the eccrine sweat and sebaceous glands., Conclusions: Selected human hair and epithelial keratins are modulated in situ. This may be relevant to explain hair shaft growth-promoting effects of TRH. Our pilot study suggests that the neuroendocrine controls that regulate the expression of human keratins deserve more systematic exploration and that these may be harnessed therapeutically., (© 2013 The Authors BJD © 2013 British Association of Dermatologists.)

The cytocompatibility, in vivo biodegradation and wound healing of keratin biomaterials were investigated. For the purposes, three groups of keratin scaffolds were fabricated by freeze-drying reduced solutions at 2 wt.%, 4 wt.% and 8 wt.% keratins extracted from human hairs. These scaffolds exhibited evenly distributed high porous structures with pore size of 120-220 μm and the porosity >90%. NIH3T3 cells proliferated well on these scaffolds in culture lasting up to 22 days. Confocal micrographs stained with AO visually revealed cell attachment and infiltration as well as scaffold architectural stability. In vivo animal experiments were conducted with 4 wt.% keratin scaffolds. Early degradation of subcutaneously implanted scaffolds occurred at 3 weeks in the outermost surface, in concomitant with inflammatory response. At 5 weeks, the overall porous structure of scaffolds severely deteriorated while the early inflammatory response in the outermost surface obviously subsided. A faster keratin biodegradation was observed in repairing full-thickness skin defects. Compared with the blank control, keratin scaffolds gave rise to more blood vessels at 2 weeks and better complete wound repair at 3 weeks with a thicker epidermis, less contraction and newly formed hair follicles. These preliminary results suggest that human hair keratin scaffolds are promising dermal substitutes for skin regeneration., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Historical evidence documents mass migration from Ireland to London during the period of the Great Irish Famine of 1845-52. The rural Irish were reliant on a restricted diet based on potatoes but maize, a C(4) plant, was imported from the United States of America in 1846-47 to mitigate against Famine. In London, Irish migrants joined a population with a more varied diet. To investigate and characterize their diet, carbon and nitrogen isotope ratios were obtained from bone collagen of 119 and hair keratin of six individuals from Lukin Street cemetery, Tower Hamlets (1843-54), and bone collagen of 20 individuals from the cemetery at Kilkenny Union Workhouse in Ireland (1847-51). A comparison of the results with other contemporaneous English populations suggests that Londoners may have elevated δ(15) N compared with their contemporaries in other cities. In comparison, the Irish group have lower δ(15) N. Hair analysis combined with bone collagen allows the reconstruction of perimortem dietary changes. Three children aged 5-15 years from Kilkenny have bone collagen δ(13) C values that indicate consumption of maize (C(4)). As maize was only imported into Ireland in quantity from late 1846 and 1847, these results demonstrate relatively rapid bone collagen turnover in children and highlight the importance of age-related bone turnover rates, and the impact the age of the individual can have on studies of short-term dietary change or recent migration. Stable light isotope data in this study are consistent with the epigraphic and documentary evidence for the presence of migrants within the London cemetery., (Copyright © 2012 Wiley Periodicals, Inc.)

Keratin-associated proteins (KAPs) are major structural components of hair and wool fibres, and play a critical role in determining the properties of the fibre. While over 100 KAP genes that have been grouped into 27 KAP families have been identified in mammals, most homologues remain unidentified in sheep. A BLAST search of the Ovine Genome Assembly v2.0 using a human KRTAP24-1 coding sequence (NM_001085455), identified a putative ovine KAP24-1 gene clustered with six other known KAP genes on chromosome 1. The KAP24-1 gene was amplified from the genomic DNA of 260 New Zealand Romney-cross sheep and stem-loop conformational polymorphism (SLCP) analysis of the amplicons revealed four unique banding-patterns, representing four different DNA sequences. These sequences were not closely homologous with any known ovine KRTAP and the highest similarity was with KRTAP24-1 sequences from humans, cattle, dog, pig, Sumatran orangutan and northern white-cheeked gibbon. This suggests that the sequences were allelic variants of ovine KRTAP24-1. Among these four sequences, seven nucleotide substitutions in the coding region were identified and four of the substitutions were non-synonymous. The putative ovine KAP24-1 polypeptide consisted of 252 amino acids. While probably belonging to the high-sulphur KAP group, the polypeptide had a moderate level of cysteine, but a high content of serine and tyrosine. The polypeptide possesses two putative N-glycosylation sites and a number of residues that may be O-glycosylated and/or phosphorylated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Keratin is a protein in the intermediate filament family and the key component of hair, nail, and skin. Here we report a bottom-up atomistic model of the keratin dimer, using the complete human keratin type k35 and k85 amino acid sequence. A detailed analysis of geometric and mechanical properties through full-atomistic simulation with validation against experimental results is presented. We introduce disulfide cross-links in a keratin tetramer and compare the mechanical behavior of the disulfide bonded systems with a system without disulfide bonds. Disulfide bond results in a higher strength (20% increase) and toughness (49% increase), but the system loses α-helical structures under loading, suggesting that disulfide bonds play a significant role in achieving the characteristic mechanical properties of trichocyte α-keratin. Our study provides general insight into the effect of disulfide cross-link on mechanical properties. Moreover, the availability of an atomistic model of this protein opens the possibility to study the mechanical properties of hair fibrils and other fibers from a bottom-up perspective.

Hair keratin is a composite structure in which intermediate filaments (IF) are embedded in a protein matrix. During the early stages of development in the hair follicle the redox potential is such that the cysteine residues in the IF are maintained in a reduced form. However, at a late stage of development the redox potential changes to produce an oxidizing environment and the IF undergo a structural transition involving both molecular slippage and radial compaction. In our earlier study the changes in the molecular parameters were estimated from knowledge of the sites of artificially induced crosslinks, and it was noted that the changes in these parameters realigned many of the cysteine residues to positions more favorable to disulfide bond formation. As the energy involved in the formation of disulfide bonds is much greater than that of hydrogen bonds or van der Waals interactions the structural transition is likely to be dominated by the requirement that the bonded cysteine residues occur at closely equivalent axial positions. This criterion was used in the present study to obtain more precise values for the molecular parameters in the oxidized fiber than has hitherto been possible. A comparison of the sequences of hair keratins and epidermal keratins suggests that the slippage observed in trichocyte IF during keratinization does not occur in epidermal IF., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Molecular dynamics simulations of a keratin/peptide complex have been conducted to predict the binding affinity of four different peptides toward human hair. Free energy calculations on the peptides' interaction with the keratin model demonstrated that electrostatic interactions are believed to be the main driving force stabilizing the complex. The molecular mechanics-Poisson-Boltzmann surface area methodology used for the free energy calculations demonstrated that the dielectric constant in the protein's interior plays a major role in the free energy calculations, and the only way to obtain accordance between the free energy calculations and the experimental binding results was to use the average dielectric constant., (Copyright © 2012 Wiley Periodicals, Inc.)

Human hair is principally composed of hair keratins and keratin-associated proteins (KAPs) that form a complex network giving the hair its rigidity and mechanical properties. However, during their growth, hairs are subject to various treatments that can induce irreversible damage. For a better understanding of the human hair protein structures, proteomic mass spectrometry (MS)-based strategies could assist in characterizing numerous isoforms and posttranslational modifications of human hair fiber proteins. However, due to their physicochemical properties, characterization of human hair proteins using classical proteomic approaches is still a challenge. To address this issue, we have used two complementary approaches to analyze proteins from the human hair cortex. The multidimensional protein identification technology (MudPit) approach allowed identifying all keratins and the major KAPs present in the hair as well as posttranslational modifications in keratins such as cysteine trioxidation, lysine, and histidine methylation. Then two-dimensional gel electrophoresis coupled with MS (2-DE gel MS) allowed us to obtain the most complete 2-DE gel pattern of human hair proteins, revealing an unexpected heterogeneity of keratin structures. Analyses of these structures by differential peptide mapping have brought evidence of cleaved species in hair keratins and suggest a preferential breaking zone in α-helical segments., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Single-compound analysis of stable or radio-isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compounds of interest.Here, we describe three complementary preparative HPLC procedures suitable for separating and isolating single amino acids from bone collagen or hair keratin with minimal isotopic contamination. Using preparative reversed-phase, ion-pair, or mixed-mode chromatography of underivatized amino acids in aqueous mobile phases, single amino acids can be isolated and further analyzed using mass spectrometric techniques.

Keratins (Ks) consist of central α-helical rod domains that are flanked by non-α-helical head and tail domains. The cellular abundance of keratins, coupled with their selective cell expression patterns, suggests that they diversified to fulfill tissue-specific functions although the primary structure differences between them have not been comprehensively compared. We analyzed keratin sequences from many species: K1, K2, K5, K9, K10, K14 were studied as representatives of epidermal keratins, and compared with K7, K8, K18, K19, K20 and K31, K35, K81, K85, K86, which represent simple-type (single-layered or glandular) epithelial and hair keratins, respectively. We show that keratin domains have striking differences in their amino acids. There are many cysteines in hair keratins but only a small number in epidermal keratins and rare or none in simple-type keratins. The heads and/or tails of epidermal keratins are glycine and phenylalanine rich but alanine poor, whereas parallel domains of hair keratins are abundant in prolines, and those of simple-type epithelial keratins are enriched in acidic and/or basic residues. The observed differences between simple-type, epidermal and hair keratins are highly conserved throughout evolution. Cysteines and histidines, which are infrequent keratin amino acids, are involved in de novo mutations that are markedly overrepresented in keratins. Hence, keratins have evolutionarily conserved and domain-selectively enriched amino acids including glycine and phenylalanine (epidermal), cysteine and proline (hair), and basic and acidic (simple-type epithelial), which reflect unique functions related to structural flexibility, rigidity and solubility, respectively. Our findings also support the importance of human keratin 'mutation hotspot' residues and their wild-type counterparts.

Cultural heritage contains a large number of precious proteinaceous specimens, such as wool and silk textiles, leather objects, paper, paint, coatings, binders (and associated adhesives), etc. To minimize the degradation of and to preserve these artifacts, it is desirable to understand the fundamental factors that cause their degradation, to identify the deterioration markers that determine their degradation stage and their age, and to use technologies that can provide this information rapidly while consuming a minimal amount of sample. There are several forces that cause protein degradation, including amino acid racemization, protein deamidation, and protein truncation. The purpose of this paper is to study protein deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for high-throughput dating of museums wool specimens. For proof of concept, several well-dated sheep's wool textiles from museum collections were analyzed. For wool samples aged from the present to ~400 years ago, the deamidation of two asparagine-containing peptides obtained from the tryptic digest of sheep wool were found to behave linearly in time, indicating that they could act as a potential biomarker of aging for wool samples., (Copyright © 2011 John Wiley & Sons, Ltd.)

The limited source of human nail plate for studying drug permeation inspired us to develop a nail plate model made of human hair keratin. The manufacturing process consisted of keratin extraction, dialysis, molding, solvent evaporation, and curing, producing a water-resistant film. The permeability of the film was examined using three markers: sodium fluorescein, rhodamine B, and fluorescein isothiocyanate-dextran as water-soluble, lipid-soluble, and large molecule models, respectively. Bovine hoof was used for comparison. First investigation showed that keratin films (thickness 120 μm) resembled hooves (thickness 100 μm) except that these films were more permeable to rhodamine B compared with hooves (1.8-fold, p<0.01). Subsequent investigations using ungual penetration enhancers (urea, thioglycolic acid, and papain) showed that keratin films were generally more susceptible than hooves. This study revealed that the produced keratin film offers a possibility as a human nail plate substitute. However, inclusion of the penetration enhancer must be carefully interpreted., (Copyright © 2011 Elsevier B.V. All rights reserved.)

It is well known that N(2) in the ion source of a mass spectrometer interferes with the CO background during the δ(18)O measurement of carbon monoxide. A similar problem arises with the high-temperature conversion (HTC) analysis of nitrogenous O-bearing samples (e.g. nitrates and keratins) to CO for δ(18)O measurement, where the sample introduces a significant N(2) peak before the CO peak, making determination of accurate oxygen isotope ratios difficult. Although using a gas chromatography (GC) column longer than that commonly provided by manufacturers (0.6 m) can improve the efficiency of separation of CO and N(2) and using a valve to divert nitrogen and prevent it from entering the ion source of a mass spectrometer improved measurement results, biased δ(18)O values could still be obtained. A careful evaluation of the performance of the GC separation column was carried out. With optimal GC columns, the δ(18)O reproducibility of human hair keratins and other keratin materials was better than ± 0.15 ‰ (n=5; for the internal analytical reproducibility), and better than ± 0.10 ‰ (n=4; for the external analytical reproducibility)., (Copyright © 2011 John Wiley & Sons, Ltd.)

Trichocyte intermediate filament protein (IFP) is a heterodimeric complex that plays a pivotal role in the hair shaft for its mechanical strength, hair shape, and so on. Trichocyte IFP consists of acidic-type IFP and basic-type IFP, and the well-studied supramolecular assembly process of the complex occurs via the following steps: dimer formation, tetramer formation, formation of the lateral 32mer, and the elongation of the 32mer. Among these interactions, only the dimer formation, owing to coiled-coil interaction, has been described in detail; the nature of other interactions remains unspecified. For each assembly step, we report interaction isotherms obtained by means of isothermal titration calorimetry at various urea and NaCl concentrations. Decreasing the urea concentration generally promotes protein refolding, and we therefore expected to observe endothermic interactions owing to the refolding process. However, exothermic interactions were observed at 4 and 2 M urea, along with various characteristic endothermic interactions at the other urea concentrations as well as NaCl titration. The thermal responses described herein enabled us to analyze the protein supramolecular assembly process in a stepwise manner., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Examination of very long hair (length > 2.4 m) using a large range of evaluation methods including physical, chemical, biochemical and microscopic techniques has enabled to attain a detailed understanding of natural ageing of human hair keratin fibres. Scrutinizing hair that has undergone little or no oxidative aggression--because of the absence of action of chemical agents such as bleaching or dyeing--from the root to the tip shows the deterioration process, which gradually takes place from the outside to the inside of the hair shaft: first, a progressive abrasion of the cuticle, whilst the cortex structure remains unaltered, is evidenced along a length of roughly 1 m onwards together with constant shine, hydrophobicity and friction characteristics. Further along the fibre, a significant damage to cuticle scales occurs, which correlates well with ceramides and 18-Methyl Eicosanoic Acid (18-MEA) decline, and progressive decrease in keratin-associated protein content. Most physical descriptors of mechanical and optical properties decay significantly. This detailed description of natural ageing of human hair fibres by a fine analysis of hair components and physical parameters in relationship with cosmetic characteristics provides a time-dependent 'damage scale' of human hair, which may help in designing new targeted hair care formulations.