Valorization of feather waste in Brazil: structure, methods of extraction, and applications of feather keratin.

This systematic review presents the potential of using feather waste as a ß-keratin source, including the Brazilian scenario in the generation of this byproduct. The structure and properties of α- and ß-keratin, the methods commonly reported to extract keratin from poultry feathers, and applications of feather keratin-based materials are also covered in this review. The literature search for poultry production data in Brazil was conducted for the last 2 years, for the period 2021-2022. A broad literature search for extraction methods and applications of feather keratin was done for the period 2001-2022. The poultry industry is one of the largest sectors of the food industry, and Brazil was the third-largest world producer of chicken meat with more than six billion chickens slaughtered in 2021. Poultry feathers constitute about 7% weight of broilers; thus, it can be estimated that about one million tons of poultry feathers were generated in Brazil in 2021, and the improper disposal of this byproduct contributes to environmental problems and disease transmission. The most common method of reusing feathers is the production of feather meal. From economic and environmental points of view, it is advantageous to develop processes to add value to this byproduct, including the extraction of keratin. Among natural biodegradable polymers, keratin-based materials have revolutionized the field of biomaterials due to their biocompatibility and biodegradability, allowing their application in biomedical, pharmaceutical, chemical, and engineering areas.

Preservation of corneous ß-proteins in Mesozoic feathers.

Fossil proteins are valuable tools in evolutionary biology. Recent technological advances and better integration of experimental methods have confirmed the feasibility of biomolecular preservation in deep time, yielding new insights into the timing of key evolutionary transitions. Keratins (formerly α-keratins) and corneous ß-proteins (CBPs, formerly ß-keratins) are of particular interest as they define tissue structures that underpin fundamental physiological and ecological strategies and have the potential to inform on the molecular evolution of the vertebrate integument. Reports of CBPs in Mesozoic fossils, however, appear to conflict with experimental evidence for CBP degradation during fossilization. Further, the recent model for molecular modification of feather chemistry during the dinosaur-bird transition does not consider the relative preservation potential of different feather proteins. Here we use controlled taphonomic experiments coupled with infrared and sulfur X-ray spectroscopy to show that the dominant ß-sheet structure of CBPs is progressively altered to α-helices with increasing temperature, suggesting that (α-)keratins and α-helices in fossil feathers are most likely artefacts of fossilization. Our analyses of fossil feathers shows that this process is independent of geological age, as even Cenozoic feathers can comprise primarily α-helices and disordered structures. Critically, our experiments show that feather CBPs can survive moderate thermal maturation. As predicted by our experiments, analyses of Mesozoic feathers confirm that evidence of feather CBPs can persist through deep time.

General and specific microscopic characteristics of the dorsal tail scales and the spines of the crest in the tuatara Sphenodon pucntatus (Reptilia; Rhynchocephalia; Sphenodontidae).

Dorsal crest scales and those of the tail spines of the tuatara (Sphenodon punctatus) represent different specializations involved in display and protection. Erection of the dorsal crest occurs in males during combat and courtship, but tail spines are not noticeably involved in these activities. In both scale derivatives corneous beta proteins (CBPs, formerly called beta-keratins) and intermediate filaments keratins (IFKs) were determined by immunolabelling. The dermis is dense with few sparse fibrocytes surrounded by collagen bundles, the latter rather randomly oriented in the crest scales. In the tail ridge scales banded collagen I fibrils form more regular, orthogonally aligned bundles of alternating layers with connections to the basal epidermal membrane. A conglomerate of dermal melanonophores and iridophores is present under the epidermis. The iridophores are the likely origin of the whitish colour of the crest. The epidermis shows a thicker beta-layer with serrated/indented corneocytes in the tail scales while the beta layer is reduced in the crest but contains CBPs. A relatively thick mesos layer is present in both scale derivatives, especially in the crest where its role, aside from limiting transpiration, is not known. The alpha-layer is formed by corneocytes with irregular perimeter and sparse desmosomal remnants. The high labelling intensity for CBPs in the beta-layer disappears in the mesos layer but occurs, albeit strongly reduced, in the alpha-layer as in the other body scales. The take-home message is that the dense dermis and its apical beta-layer strengthen mechanically the ridge spines while the crest is mainly supported by the firm but pliable and less dense or regular dermis.

Beta-keratins of turtle shell are glycine-proline-tyrosine rich proteins similar to those of crocodilians and birds.

This study presents, for the first time, sequences of five beta-keratin cDNAs from turtle epidermis obtained by means of 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The deduced amino acid sequences correspond to distinct glycine-proline-serine-tyrosine rich proteins containing 122-174 amino acids. In situ hybridization shows that beta-keratin mRNAs are expressed in cells of the differentiating beta-layers of the shell scutes. Southern blotting analysis reveals that turtle beta-keratins belong to a well-conserved multigene family. This result was confirmed by the amplification and sequencing of 13 genomic fragments corresponding to beta-keratin genes. Like snake, crocodile and avian beta-keratin genes, turtle beta-keratins contain an intron that interrupts the 5'-untranslated region. The length of the intron is variable, ranging from 0.35 to 1.00 kb. One of the sequences obtained from genomic amplifications corresponds to one of the five sequences obtained from cDNA cloning; thus, sequences of a total of 17 turtle beta-keratins were determined in the present study. The predicted molecular weight of the 17 different deduced proteins range from 11.9 to 17.0 kDa with a predicted isoelectric point of 6.8-8.4; therefore, they are neutral to basic proteins. A central region rich in proline and with beta-strand conformation shows high conservation with other reptilian and avian beta-keratins, and it is likely involved in their polymerization. Glycine repeat regions, often containing tyrosine, are localized toward the C-terminus. Phylogenetic analysis shows that turtle beta-keratins are more similar to crocodilian and avian beta-keratins than to those of lizards and snakes.

Localization of Alpha-Keratin and Beta-Keratin (Corneous Beta Protein) in the Epithelium on the Ventral Surface of the Lingual Apex and Its Lingual Nail in the Domestic Goose (Anser Anser f. domestica) by Using Immunohistochemistry and Raman Microspectroscopy Analysis.

The epithelium of the ventral surface of the apex of the tongue in most birds is specified by the presence of the special superficial layer called lingual nail. The aim of the present study is to determine the localization of the alpha-keratin and beta-keratin (corneous beta protein) in this special epithelium in the domestic goose by using immunohistochemistry staining and the Raman spectroscopy analysis. Due to lack of commercially available antibodies to detect beta-keratin (corneous beta protein), the Raman spectroscopy was used as a specific tool to detect and describe the secondary structure of proteins. The immunohistochemical (IHC) detections reveal the presence of alpha-keratin in all layers of the epithelium, but significant differences in the distribution of the alpha-keratin in the epithelial layers appear. The staining reaction is stronger from the basal layer to the upper zone of the intermediate layer. The unique result is weak staining for the alpha-keratin in the lingual nail. Applications of the Raman spectroscopy as a complementary method not only confirmed results of IHC staining for alpha-keratin, but showed that this technique could be used to demonstrate the presence of beta-keratin (corneous beta protein). Functionally, the localization of alpha-keratin in the epithelium of the ventral surface of the lingual apex provides a proper scaffold for epithelial cells and promotes structural integrity, whereas the presence of beta-keratin (corneous beta protein) in the lingual nail, described also as exoskeleton of the ventral surface of the apex, endures mechanical stress. Anat Rec, 300:1361-1368, 2017. © 2017 Wiley Periodicals, Inc.

Distribution and characterization of keratins in the epidermis of the tuatara (Sphenodon punctatus; Lepidosauria, Reptilia).

Reptilian scales are mainly composed of alpha-and beta-keratins. Epidermis and molts from adult individuals of an ancient reptilian species, the tuatara (Sphenodon punctatus), were analysed by immunocytochemistry, mono- and bi-dimensional electrophoresis, and western blotting for alpha- and beta-keratins. The epidermis of this reptilian species with primitive anatomical traits should represent one of the more ancient amniotic epidermises available. Soft keratins (AE1- and AE3-positive) of 40-63 kDa and with isoelectric points (pI) at 4.0-6.8 were found in molts. The AE3 antibody was diffusely localised over the tonofilaments of keratinocytes. The lack of basic cytokeratins may be due to keratin alteration in molts, following corneification or enzymatic degradation of keratins. Hard (beta-) keratins of 16-18 kDa and pI at 6.8, 8.0, and 9.2 were identified using a beta-1 antibody produced against chick scale beta-keratin. The antibody also labeled filaments of beta-cells and of the mature, compact beta-layer. We have shown that beta-keratins in the tuatara resemble those of lizards and snakes, and that they are mainly basic proteins. These proteins replace cytokeratins in the pre-corneoum beta-layers, from which a hard, mechanically resistant corneoum layer is formed over scales. Beta-keratins may have both a fibrous and a matrix role in forming the hard texture of corneoum scales in this ancient species, as well as in more recently evolved reptiles.

Cornification in reptilian epidermis occurs through the deposition of keratin-associated beta-proteins (beta-keratins) onto a scaffold of intermediate filament keratins.

The isolation of genes for alpha-keratins and keratin-associated beta-proteins (formerly beta-keratins) has allowed the production of epitope-specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha-keratin range (40-70 kDa) or beta-protein range (10-30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha-proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin-associated beta-proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin-associated beta-proteins is typical for keratin-associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine-rich or sulfur-rich proteins). In turtles and crocodilians epidermis, keratin-associated beta-proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine-rich versus cysteine-glycine-rich keratin-associated beta-proteins in cells sequentially produced from the basal layer and not from the alternation of beta- with alpha-keratins. The process gives rise to Oberhäutchen, beta-, mesos-, and alpha-layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin-associated proteins (KAPs) of the epidermis, the keratin-associated beta-proteins of sauropsids are capable to form filaments of 3-4 nm which give rise to an X-ray beta-pattern as a consequence of the presence of a beta-pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates.

Ultrastructural immunolocalization of alpha-keratins and associated beta-proteins (beta-keratins) suggests a new interpretation on the process of hard and soft cornification in turtle epidermis.

The epidermis of soft-shelled and hard-shelled turtles has been compared to determine the origin of the different cornification. Immunolocalization of acidic alpha-keratin (AK2) of 45-50 kDa in tonofilaments of the epidermis in Apalone spinifera and absence in the corneous layer where desquamating corneocytes are present supports the biochemical data. Corneocytes shows a weak to absent immunolabeling for beta-proteins (formerly beta-keratins) of 14-16 kDa while sparse immunolabeled corneous granules are seen in the pre-corneous layer. In the hard-shelled turtle Pseudemys nelsonii differentiating corneocytes contain small level of acidic alpha-keratin while beta-proteins of 10-17 kDa form dense aggregates of corneous material among tonofilaments. Corneocytes do not desquamate but remain tightly connected determining an increase in thickness of the corneous layer that becomes mechanically stiff and resistant. Since both species possess beta-proteins in shelled and non-shelled areas of the epidermis the difference in hardness of the corneous layer is not due to the alternation between beta-keratin versus alpha-keratin. Mechanical resilience of the corneous layer derives from the accumulation of alpha-keratins, beta- and likely of other proteins in corneocytes of the shell in hard-shelled turtles. In the softer epidermis of hard-shelled turtles and in the soft-shelled turtle a more rapid and continuous turnover of corneocytes is present and no accumulation of beta-proteins and corneocytes takes place. It is hypothesized that the dermis derived from the carapacial ridge during development remains localized underneath the shell epidermis in hard-shelled turtles and influences the formation of the hard corneous epidermis.

Immunolocalization of keratin-associated beta-proteins in developing epidermis of lizard suggests that adhesive setae contain glycine--cysteine-rich proteins.

The localization of specific keratin-associated beta-proteins (formerly referred to as beta-keratins) in the embryonic epidermis of lizards is not known. Two specific keratin-associated beta-proteins of the epidermis, one representing the glycine-rich subfamily (HgG5) and the other the glycine-cysteine medium-rich subfamily (HgGC10), have been immunolocalized at the ultrastructural level in the lizard Anolis lineatopus. The periderm and granulated subperiderm are most immunonegative for these proteins. HgG5 is low to absent in theOberhäutchen layer while is present in the forming beta-layer, and disappears in mesos- and alpha-layers. Instead, HgGC10 is present in the Oberhäutchen, beta-, and also in the following alpha-layers, and specifically accumulates in the developing adhesive setae but not in the surrounding cells of the clear layer. Therefore, setae and their terminal spatulae that adhere to surfaces allowing these lizards to walk vertically contain cysteine-glycine rich proteins. The study suggests that, like in adult and regenerating epidermis, the HgGC10 protein is not only accumulated in cells of the beta-layer but also in those forming the alpha-layer. This small protein therefore is implicated in resistance, flexibility, and stretching of the epidermal layers. It is also hypothesized that the charges of these proteins may influence adhesion of the setae of pad lamellae. Conversely, glycine-rich beta-proteins like HgG5 give rise to the dense, hydrophobic, and chromophobic corneous material of the resistant beta-layer. This result suggests that the differential accumulation of keratin-associated beta-proteins over the alpha-keratin network determines differences in properties of the stratified layers of the epidermis of lizards.

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis.

The claw of lizards is largely composed of beta-keratins, also referred to as keratin-associated beta-proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta-keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha-keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha-keratins resembled that of beta-keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins.

Evaluating uncertainty in the calculation of non-exchangeable hydrogen fractions within organic materials.

We calculated the fraction of exchangeable hydrogen atoms in proteinaceous materials commonly analyzed for stable isotopic composition related to the region-of-origin of an animal. These included several types of alpha- and beta-keratin, and muscle tissue. We find that the fraction of H atoms in keratin available for exchange at a biologically relevant temperature (25 degrees C) averaged 9% across a range of ground organic materials, but was as high as approximately 17% in cut hair; muscle tissue has approximately 12% exchangeable H atoms. Under most analysis conditions, the difference in exchangeable fractions due to physical sample processing has a minimal effect on the calculated delta2H values of the non-exchangeable H atoms within a keratin-containing tissue (<2 per thousand). However, extreme mismatches between sample and reference material types could affect delta2H values.

Immunological characterization of a newly developed antibody for localization of a beta-keratin in turtle epidermis.

Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.