Development of a Combined Protein and Dye Extraction Approach for the Analysis of Keratin-Based Textiles.

Archaeological textiles represent precious remains from ancient culture; this is because of the historical and cultural importance of the information that can be obtained by such relics. However, the extremely complicated state of preservation of these textiles, which can be charred, partially or totally mineralized, with heavy soil or biological contamination, requires highly specialized and sensitive analytical tools to perform a comprehensive study. Starting from these considerations, the paper presents a combined workflow that provides the extraction of dyes and keratins and keratin-associated proteins in a single step, minimizing sampling while maximizing the amount of information gained. In the first phase, different approaches were tested and two different protocols were found suitable for the purpose of the unique workflow for dyes/keratin-proteins: a slightly modified urea protocol and a recently proposed new TCEP/CAA procedure. In the second step, after the extraction, different methods of cleanup and workflow for proteins and dyes were investigated to develop protocols that did not result in a loss of aliquots of the analytes of interest and to maximize the recovery of both components from the extracting solution. These protocols investigated the application of two types of paramagnetic beads, unmodified and carboxylate-coated hydrophilic magnetic beads, and dialysis and stage-tip protocols. The newly designed protocols have been applied to cochineal, weld, orchil, kermes, and indigo keratin-based dyed samples to evaluate the effectiveness of the protocols on several dye sources. These protocols, based on a single extraction step, show the possibility of investigating dyes and keratins from a unique sample of 1 mg or lesser, with respect to the thresholds of sensitivity and accuracy required in the study of textile artifacts of historical and artistic values.

Stability and cytotoxicity of DPPH inhibitory peptides derived from biodegradation of chicken feather.

The antioxidant activity and cell viability of feather hydrolysates obtained with the Bacillus licheniformis were evaluated using an in-vitro model. The results indicate that feathers-derived peptides under 3 kDa have antioxidant activity with IC50 values of 5.03 ± 0.215 mg/mL by using DPPH antioxidant assay. Although the antioxidant activity of the peptides under 3 kDa preserved after applying diverse heating (from 20 to 100 °C), they lost their activity under strongly acidic or alkaline conditions. Antioxidant activity of the mixed feather bioactive peptides (MFBPs) obtained with partial purification of peptides under 3 kDa was with IC50 amount of 0.169 mg/mL ± 0.004 using DPPH radical scavenging assay. Also, MFBPs within an amount range of from 0.0048 to 5.0 mg/mL, illustrated no cytotoxicity to gingival fibroblast blood cell lines. In light of our results, the obtained value-added peptides could be useful in different food products as a future functional ingredient with antioxidant potency.

One-Pot Process: Microwave-Assisted Keratin Extraction and Direct Electrospinning to Obtain Keratin-Based Bioplastic.

Poultry feathers are among the most abundant and polluting keratin-rich waste biomasses. In this work, we developed a one-pot microwave-assisted process for eco-friendly keratin extraction from poultry feathers followed by a direct electrospinning (ES) of the raw extract, without further purification, to obtain keratin-based bioplastics. This microwave-assisted keratin extraction (MAE) was conducted in acetic acid 70% v/v. The effects of extraction time, solvent/feathers ratio, and heating mode (MAE vs. conventional heating) on the extraction yield were investigated. The highest keratin yield (26 ± 1% w/w with respect to initial feathers) was obtained after 5 h of MAE. Waste-derived keratin were blended with gelatin to fabricate keratin-based biodegradable and biocompatible bioplastics via ES, using 3-(Glycidyloxypropyl)trimethoxysilane (GPTMS) as a cross-linking agent. A full characterization of their thermal, mechanical, and barrier properties was performed by differential scanning calorimetry, thermogravimetric analysis, uniaxial tensile tests, and water permeability measurements. Their morphology and protein structure were investigated using scanning electron microscopy and attenuated total reflection-infrared spectroscopy. All these characterizations highlighted that the properties of the keratin-based bioplastics can be modulated by changing keratin and GPTMS concentrations. These bioplastics could be applied in areas such as bio-packaging and filtration/purification membranes.

Surface analysis of ancient parchments via the EVA film: The Aleppo Codex.

The margins of several pages of the Aleppo codex have been found to be corroded and contaminated by diffuse maculae. In order to understand the origin of this decay these margins have been analysed by applying EVA (ethylene vinyl acetate plastic embedded with strong cation and anion exchangers and mixed with C8 and C18 hydrophobic resins) diskettes for harvesting surface material. The captured compounds have been eluted, digested with trypsin and analysed by nano-HPLC-MS. Three major strains of Aspergillus have been identified, namely Aspergillus fumigatus, Aspergillus pseudoglaucus, Aspergillus amstelodami, together with a lactobacillus strain and human keratins. The novelty of this investigation is that for the first time the EVA technology has been applied to ancient parchments in the absence of mechanical deformation or distortion that could be induced if there had been water exchange between the EVA diskettes and the parchment. These findings should help curators to find suitable restoration protocols for these precious documents belonging to the world Cultural Heritage.

Expression and purification of high sulfur and high glycine-tyrosine keratin-associated proteins (KAPs) for biochemical and biophysical characterization.

Keratin-associated proteins (KAPs) were identified 70 years ago in wool follicles. KAPs are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. However, detailed molecular structural information is lacking for KAPs and for KAP interactions with IFs. As a preliminary step towards their biophysical and structural characterization, we have expressed and purified a HS KAP (KAP11.1) and a HGT KAP (KAP6.1). The expression and purification of KAPs is challenging because they are cysteine-rich proteins with unusual amino acid compositions, they tend to be insoluble in isolation and are prone to forming aggregates in solution. Here we describe the high yield production of pure, soluble KAPs in a chaotrope- and detergent-free buffer. This method has the potential to be used for the overproduction of other KAPs and similar cysteine-rich proteins with high isoelectric points.

Preparation of wool follicles for proteomic studies.

A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs.

Lacticaseibacillus rhamnosus encapsulated cross-linked Keratin-Chitosan hydrogel for removal of patulin from apple juice.

In this study, we developed a hydrogel from cross-linked keratin and chitosan (KC) to remove patulin (PAT) from apple juice. We explored the potential of incorporating Lactobacillus rhamnoses into the KC hydrogel (KC-LR) and tested its effectiveness in removing PAT from simulated juice solutions and real apple juice. The KC hydrogel was developed through a dynamic disulfide cross-linking reaction. This cross-linked hydrogel network provided excellent stability for the probiotic cells, achieving 99.9 % immobilization efficiency. In simulated juice with 25 mg/L PAT, the KC and KC-LR hydrogels showed removal efficiencies of 85.2 % and 97.68 %, respectively, using 15 mg mL-1 of the prepared hydrogel at a temperature of 25 °C for 6 h. The KC and KC-LR hydrogels achieved 76.3 % and 83.6 % removal efficiencies in real apple juice systems, respectively. Notably, the encapsulated probiotics did not negatively impact the juice quality and demonstrated reusability for up to five cycles of the PAT removal process.

Purification of porcine hair keratin subunits and their immobilization for use as cell culture substrates.

We purified both the type I subunit and type II subunit of porcine hair keratin and compared their ability to form a uniform film of reconstituted keratin on a culture plate, and their effect on a model of neural cells. We observed the surface of the keratin-immobilized plate using a scanning electron microscope (SEM) and measured water contact angles to characterize the surface. We cultured PC12 cells on plates on which crude keratin, the type I subunit, or the type II subunit were immobilized. The water contact angles were slightly different from each other. The cells proliferated well on all three keratin-immobilized plates. The type II subunit showed a tendency to inhibit the differentiation of PC12 cells significantly as an extension of the cell shapes and neurite outgrowth in comparison with the crude extract and the type I subunit. The type I subunit and the type II subunit showed slight differences in cell differentiation, but not in cell proliferation.

An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis.

In this study, the possibility of keratin extraction from wool and feather by an enzymatic treatment along with a reducing agent has been investigated. The effects of different parameters, that is, enzyme loading, type of substrate and surfactant, hydrolysis time, and reducing agent concentration, have been examined in order to optimize the enzymatic hydrolysis. The optimal condition for maximum keratin extraction was attained by making use of 1 g/L sodium dodecyl sulfate (an anionic surfactant) and 2.6% (v/v) protease (Savinase), along with 8.6 and 6.4 g/L sodium hydrogen sulfite (a reducing agent) for wool and feathers, respectively, at liquor to fiber ratio of 25 mL/g for 4 hr. The obtained results indicated higher degradation of wool fiber in comparison with feathers, which might be due to the higher hydrophilic nature of the former. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns revealed that the molecular weights of the extracted proteins from wool and feather were lower than those for the untreated fibers. Scanning electron micrographs showed fibers fibrillation and degradation upon enzymatic treatment. Besides, Fourier-transform infrared (FTIR) spectra indicated no evident changes in the chemical structure of the hydrolyzed fibers. However, wool and feather remainders were mostly composed of α-helix and ß-sheets conformations, respectively.

Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium.

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.

Biopsy of a 5 mm cystic lesion on the right heel of a 48-year-old woman.

An excisional biopsy of an asymptomatic cystic lesion that had been present for several years on the right heel of a 48-year-old woman revealed a subcutaneous cyst lined by ciliated columnar epithelium. On immunostaining, the epithelial cells were positive for Pan-cytokeratin (CK AE 1/3), estrogen receptor (ER) and progesterone receptor (PR), but negative for carcinoembryonic antigen (CEA), suggesting Mullerian type of epithelium. Cutaneous ciliated cyst of Mullerian type occurs almost exclusively on the lower extremity of premenopausal women. The lesion is benign and excision is curative.

Newer guar gum ester/chicken feather keratin interact films for tissue engineering.

This work intends to synthesis newer guar gum indole acetate ester and design film scaffolds based on protein-polysaccharide interactions for tissue engineering applications. Guar gum indole acetate(GGIA) was synthesized for the first time from guar gum in presence of aprotic solvent activated hofmeister ions. The newer biopolymer was fully characterized in FT-IR,13C NMR, XRD and TGA analysis. High DS (Degree of Substitution, DS = 0.61) GGIA was cross-linked with hydrolyzed keratin, extracted from chicken feather wastes. Films were synthesized from different biopolymer ratios and the surface chemistry appeared interesting. Physicochemical properties for GGIA-keratin association were notable. Fully bio-based films were non-cytotoxic and exhibited excellent biocompatibility for human dermal fibroblast cell cultivations. The film scaffold showed 63% porosity and the recorded tensile strength at break was 6.4 MPa. Furthermore, the standardised film exerted superior antimicrobial activity against both the Gram-positive and Gram-negative bacteria. MICs were recorded at 130 µg/mL and 212 µg/mL for E. coli and S. aureus respectively. In summary, GGIA-keratin film scaffolds represented promising platforms for skin tissue engineering applications.

Microwave-assisted extraction of human hair proteins.

The effectiveness of microwave-assisted extraction of proteins from human hair samples was evaluated. Extractions were performed from 2-mg hair samples in an extraction solution consisting of 25 mM Tris-HCl (pH 8.5), 2.6 M thiourea, 5 M urea, and 5% mercaptoethanol. During extraction, samples were exposed to microwave radiation (600 W) for a specified incubation period (5-120 min). The extraction efficiency of samples that had been incubated for 60 min was similar to that of samples that had been heated at 50°C for 24 h using the conventional Shindai method.

A new approach to epithelial-mesenchymal transition diagnostics in epithelial tumors: double immunofluorescent staining and flow cytometry.

A new method of double immunofluorescent staining for flow cytometry has been created to evaluate quantitative expression of mesenchymal protein vimentin only in epithelial cells of a solid tumor that is a mix of different origin cells. De novo vimentin expression is strongly associated with epithelial-mesenchymal transition and therefore is a metastatic potential marker of epithelial tumor cells. In comparison with semiquantitative available methods, the proposed one has several advantages, such as the accurate measurement of the marker's expression, and minimization of spatial and temporal tumor heterogeneity. Clinical validation of the method has revealed inverse correlation between the quantitative index of epithelial-mesenchymal transition level and progression-free survival using Kaplan-Meier curves and the COX proportional hazards ratio in 32 ovarian cancer patients.

Design and in silico modeling of Indoloquinoxaline incorporated keratin nanoparticles for modulation of glucose metabolism in 3T3-L1 adipocytes.

The following study was done to assess the glucose utilizing efficiency of Indoloquinoxaline derivative incorporated keratin nanoparticles (NPs) in 3T3-L1 adipocytes. Indoloquinoxaline derivative had wide range of biological activities including antidiabetic activity. In this view, Indoloquinoxaline moiety containing N, N-dimethyl (3-fluoro-6H-indolo [3,2-b] quinoxalin-6-yl) methanamine compound was designed and synthesized, and further it is incorporated into keratin nanoparticles. The formulated NPs, drug entrapment efficiency, releasing capacity, stability, and physicochemical properties were characterized by various spectral analyzer and obtained results of characterizations were confirmed the properties of NPs. The analysis of mechanism underlying the glucose utilization of NPs was examined through molecular docking with identified target, and observed in silico study reports shown strong interaction of NPs in the binding pockets of AMPK and PTP1B. Based on the in silico screening, the formulated NPs was performed for in vitro cellular viability and glucose uptake studies on 3T3-L1 adipocytes. Interestingly, 40 µg of NPs displayed 78.2 ± 2.76% cellular viability, and no cell death was observed at lower concentrations. Further, the concentration dependent glucose utilization was observed at different concentrations of NPs in 3T3-L1 adipocytes. The results of NPs (40 µg) on glucose utilization have revealed eminent result 58.56 ± 4.54% compared to that of Metformin (10 µM) and Insulin (10 µM). The identified results clearly indicated that Indoloquinoxaline derivative incorporated keratin NPs significantly increased glucose utilization efficiency and protect the cells against the insulin resistance.

Identification of the Ovine Keratin-Associated Protein 2-1 Gene and Its Sequence Variation in Four Chinese Sheep Breeds.

Keratin-associated proteins are important components of wool fibers. The gene encoding the high-sulfur keratin-associated protein 2-1 has been described in humans, but it has not been described in sheep. A basic local alignment search tool nucleotide search of the Ovine Genome Assembly version 4.0 using a human keratin-associated protein 2-1 gene sequence revealed a 399-base pair open reading frame, which was clustered among nine previously identified keratin-associated protein genes on chromosome 11. Polymerase chain reaction-single strand conformation polymorphism analysis revealed four different banding patterns, with these representing four different sequences (A-D) in Chinese sheep breeds. These sequences had the highest similarity to human keratin-associated protein 2-1 gene, suggesting that they represent variants of ovine keratin-associated protein 2-1 gene. Nine single nucleotide variations were detected in the gene, including one non-synonymous nucleotide substitution. Differences in variant frequencies between fine-wool sheep breeds and coarse-wool sheep breeds were detected. The gene was found to be expressed in various tissues, with the highest expression level in skin, and moderate expression levels in heart and lung tissue. These results reveal that the ovine keratin-associated protein 2-1 gene is variable and suggest the gene might affect variation in mean fiber diameter.

Enzymatic Extraction of Bioactive and Self-Assembling Wool Keratin for Biomedical Applications.

Keratin is widely recognized as a high-quality renewable protein resource for biomedical applications. Despite their extensive existence, keratin resources such as feathers, wool, and hair exhibit high stability and mechanical properties because of their high disulfide bond content. Consequently, keratin extraction is challenging and its application is greatly hindered. In this work, a biological extraction strategy is proposed for the preparation of bioactive keratin and the fabrication of self-assembled keratin hydrogels (KHs). Based on moderate and controlled hydrolysis by keratinase, keratin with a high molecular weight of approximately 45 and 28 kDa that retain its intrinsic bioactivities is obtained. The keratin products show excellent ability to promote cell growth and migration and are conferred with significant antioxidant ability because of their intrinsically high cysteine content. In addition, without the presence of any cross-linking agent, the extracted keratin can self-assemble into injectable hydrogels. The KHs exhibit a porous network structure and 3D culture ability, showing potential in promoting wound healing. This enzyme-driven keratin extraction strategy opens up a new approach for the preparation of keratin that can self-assemble into injectable hydrogels for biomedical engineering.

Methods of keratin extraction from poultry feathers and their effects on antioxidant activity of extracted keratin.

The antioxidant activities of extracted keratin from chicken feathers using different chemical treatments including 2-mercaptoethanol (TME), sodium sulfite (SS), and sodium dodecyl sulfate (SDS) were investigated using an in-vitro study. The results displayed that all of these methods have antioxidant potency. However, the reduction of keratin with SS displayed the best antioxidant potency with IC50 value of 0.533 ± 0.061 mg/mL using DPPH radical scavenging assay with a porous surface morphology as monitored by scanning electron microscopy (SEM). Compared to raw feathers, all extracted keratins had lower decomposition temperatures and melting points investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) respectively. Fourier-transform infrared spectroscopy (FTIR) confirmed that the extracted keratins maintained the most amides bonds in the procedure of alkaline extraction.

Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins.

BACKGROUND: Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 ("A") domain and a unique "B" domain with a nuclear localization sequence. OBJECTIVE: To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal "AB" domain and compare it to other S100 fused-type proteins. METHODS: We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. RESULTS: Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. CONCLUSION: The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.

Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments.

We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers averaging 33 and 35 kdalton/nm, respectively, which probably represent mature IF. Taken together with earlier results on IF of other subclasses, these results indicate that the average linear density of IF scales according to the average mass of their constituent subunits, both for "minimal form" and for mature IF. As underlying mechanism for this homology, we propose that the fundamental building-blocks of all these IF contain a common structural element whose packing within the various IF is likewise conserved and which specifies the overall structure. The variable amounts of mass in the nonconserved moieties account for the observed proportionality. This scheme fits with amino acid sequence data for several IF subunits that have revealed, as a likely candidate for the common element, an essentially conserved alpha-helical domain, contrasting with the highly variable sequences of their non-alpha-helical terminal domains.