The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress.

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central "rod" region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.

Predictive Potential of MALDI-TOF Analyses for Wine and Brewing Yeast.

The potential of MALDI-TOF profiling for predicting potential applications of yeast strains in the beverage sector was assessed. A panel of 59 commercial yeasts (47 wine and 12 brewing yeasts) was used to validate the concept whereby 2 culture media (YPD agar and YPD broth), as well as two mass ranges m/z 500-4000 and m/z 2000-20,000, were evaluated for the best fit. Three machine learning-based algorithms, PCA, MDS, and UMAP, in addition to a hierarchical clustering method, were employed. Profiles derived from broth cultures yielded more peaks, but these were less well-defined compared with those from agar cultures. Hierarchical clustering more clearly resolved different species and gave a broad overview of potential strain utility, but more nuanced insights were provided by MDS and UMAP analyses. PCA-based displays were less informative. The potential of MALDI-TOF proteomics in predicting the utility of yeast strains of commercial benefit is supported in this study, provided appropriate approaches are used for data generation and analysis.

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

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

From Natural Xanthones to Synthetic C-1 Aminated 3,4-Dioxygenated Xanthones as Optimized Antifouling Agents.

Biofouling, which occurs when certain marine species attach and accumulate in artificial submerged structures, represents a serious economic and environmental issue worldwide. The discovery of new non-toxic and eco-friendly antifouling systems to control or prevent biofouling is, therefore, a practical and urgent need. In this work, the antifouling activity of a series of 24 xanthones, with chemical similarities to natural products, was exploited. Nine (1, 2, 4, 6, 8, 16, 19, 21, and 23) of the tested xanthones presented highly significant anti-settlement responses at 50 µM against the settlement of mussel Mytilus galloprovincialis larvae and low toxicity to this macrofouling species. Xanthones 21 and 23 emerged as the most effective larval settlement inhibitors (EC50 = 7.28 and 3.57 µM, respectively). Additionally, xanthone 23 exhibited a therapeutic ratio (LC50/EC50) > 15, as required by the US Navy program attesting its suitability as natural antifouling agents. From the nine tested xanthones, none of the compounds were found to significantly inhibit the growth of the marine biofilm-forming bacterial strains tested. Xanthones 4, 6, 8, 16, 19, 21, and 23 were found to be non-toxic to the marine non-target species Artemia salina (<10% mortality at 50 µM). Insights on the antifouling mode of action of the hit xanthones 21 and 23 suggest that these two compounds affected similar molecular targets and cellular processes in mussel larvae, including that related to mussel adhesion capacity. This work exposes for the first time the relevance of C-1 aminated xanthones with a 3,4-dioxygenated pattern of substitution as new non-toxic products to prevent marine biofouling.

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep.

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, gamma-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the ß-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit ß (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult ß-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.

The influence of growth conditions on MALDI-TOF MS spectra of winemaking yeast: implications for industry applications.

Previous studies have shown MALDI-TOF MS to be a powerful tool in wine yeast identification and potential prediction of application. However, it is also established that substrate composition influences protein expression, but the degree to which this may affect MALDI-TOF spectra (and analytical results thereof) has not been fully explored. To further inform assay optimisation, the influence on MALDI-TOF spectra was determined using eight Saccharomyces strains of diverse origins cultivated on grape juices from Pinot Noir and Chardonnay varieties, synthetic grape juice, and laboratory-grade artificial culture media (YPD broth and agar). Our results demonstrated significant influences of culture media on strain MALDI-TOF spectra. Yeast culture on YPD agar is recommended for taxonomic studies, with YPD broth culture of S. cerevisiae offering improved intra-subspecific differentiation Furthermore, our data supported a correlation between MALDI spectra and the potential industrial application of individual yeast strains.

Application of MALDI-TOF analysis to reveal diversity and dynamics of winemaking yeast species in wild-fermented, organically produced, New Zealand Pinot Noir wine.

Rapid yeast identification is of particular importance in monitoring wine fermentation and assessing strain application in winemaking. We used MALDI-TOF MS analysis supported by 26 S rRNA gene sequence analysis and Saccharomyces-specific PCR testing to differentiate reference and field strains recovered from organic wine production facilities in Waipara, New Zealand, in which Pinot Noir wine was produced by spontaneous fermentations in the vineyard and in the winery. Strains were isolated from each of four key stages of each ferment to evaluate changes in taxonomic diversity. MALDI-TOF MS analysis was confirmed as an excellent yeast identification method, with even closely related Saccharomyces species readily distinguished. A total of 13 indigenous species belonging to eight genera were identified from Pinot Noir ferments, with taxonomic diversity generally reducing as fermentation progressed. However, differences between the taxa recovered were observed between the vineyard and winery ferments, despite the grapes used being from the same batch. Furthermore, some consistent proteomic differences between strains of S. cerevisiae, Hanseniasporum uvarum, Candida californica, Pichia membranifaciens and Starmerella bacillaris correlated with the different fermentation systems used. The high speed, low cost, taxonomic resolution and ability to characterise subtle changes in phenotype that may result from variations in environmental conditions makes MALDI-TOF analysis an attractive tool for further and wider applications in the wine industry. Such applications may include monitoring wine fermentation to actively support the consistency of high-quality wine products, and potentially for the development of such products too.

Domestic animal proteomics in the 21st century: A global retrospective and viewpoint analysis.

Animal production and health are of significant economic importance, particularly regarding the world food supply. Animal and veterinary sciences have evolved immensely in the past six decades, particularly in genetics, nutrition, housing, management and health. To address major challenges such as those posed by climate change or metabolic disorders, it is of utmost importance to use state-of-the-art research tools. Proteomics and the other post-genomic tools (transcriptomics or metabolomics) are among them. Proteomics has experienced a considerable development over the last decades. This brought developments to different scientific fields. The use and adoption of proteomics tools in animal and veterinary sciences has some limitations (database availability or access to proteomics platforms and funding). As a result, proteomics' use by animal science researchers varies across the globe. In this viewpoint article, we focus on the developments of domestic animal proteomics over the last decade in different regions of the globe and how the researchers have coped with such challenges. In the second part of the article, we provide examples of funding, educational and laboratory establishment initiatives designed to foster the development of (animal-based) proteomics. International scientific collaboration is a definitive and key feature in the development and advancement of domestic animal proteomics. SIGNIFICANCE: Animal production and health are very important for food supply worldwide particularly as a source of proteinaceous foods. Animal and veterinary sciences have evolved immensely in the last decades. In order to address the major contemporary challenges facing animal and veterinary sciences, it is of utmost importance to use state-of-the-art research tools such as Proteomics and other Omics. Herein, we focus on the major developments in domestic animal proteomics worldwide during the last decade and how different regions of the world have used the technology in this specific research field. We address also major international efforts aiming to increase the research output in this area and highlight the importance of international cooperation to address specific problems inherent to domestic animal proteomics.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

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.

The wool proteome and fibre characteristics of three distinct genetic ovine breeds from Portugal.

Wool properties and commodity value vary considerably between breeds. In Portugal, three major ovine groups exist: Churros, Bordaleiros and Merinos. This work studies the effect of the ovine genotype on the wool proteome of such groups. Wool was collected from 15 ewes/breed and genetic groups: Churra da Terra Quente (CTQ) or Churro, Serra da Estrela (SE) or Bordaleiro and Merino Branco (MB) or Merino. Proteins were extracted and subjected to label-free proteomics analysis. A total of 50 keratinous protein groups were identified in all the samples, divided into type I and II keratins and the keratin associated proteins: high-glycine-tyrosine proteins, ultra-high sulphur proteins and high-sulphur proteins. Major differences were found between MB and CTQ with respect to K75 and K38, both medullar proteins and to a lesser extent between SE and CTQ suggesting that these might be good markers for this trait in wool. Partial least squares discriminatory analysis proved MB to be readily distinguishable from the other two breeds. Further differences were noted in keratin associated protein levels between the three breeds, normally an indicator of higher levels of orthocortex and also their relationship to high curvature, high crimp fibres like Merino. BIOLOGICAL SIGNIFICANCE: The ovine genetic type has strong effects on wool productivity parameters and quality traits. In this work, we compare the proteomes and the microscopical characteristics of the wool from three distinct ovine genetic types from Portugal: Merino, Bordaleiro and Churro. Important differences were found regarding keratin associated proteins and keratins K75 and K38, suggested as putative markers for quality traits in the wool proteome such as the average curvature.

An improved method for MALDI-TOF analysis of wine-associated yeasts.

Although MALDI-TOF mass spectrometric analysis has been applied to the characterization of yeast species important in winemaking, relatively few taxa have so far been examined, and the value of low mass peaks for identification has not, to our knowledge, been previously determined. We describe a modified (pre-mixing) procedure for extraction of low (m/z 500-4000) - and high (m/z 2000-20,000) mass range moieties detected by MALDI-TOF and compare it with a previously described, proposed standard method based on a dried-droplet approach. Thirty-three strains representing 21 yeast species were examined. We found our modified method consistently yielded more discriminatory peaks and a broader mass range detection than the proposed standard method for the species examined. Cluster analyses of MALDI-TOF profiles also indicated better separation between species when the pre-mixing method was used, especially where high mass features were used. The use of low mass features may be useful for strain-level discrimination.

Differences between ultrastructure and protein composition in straight hair fibres.

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

A Multi-Bioassay Integrated Approach to Assess the Antifouling Potential of the Cyanobacterial Metabolites Portoamides.

The cyclic peptides portoamides produced by the cyanobacterium Phormidium sp. LEGE 05292 were previously isolated and their ability to condition microcommunities by allelopathic effect was described. These interesting bioactive properties are, however, still underexplored as their biotechnological applications may be vast. This study aims to investigate the antifouling potential of portoamides, given that a challenge in the search for new environmentally friendly antifouling products is to find non-toxic natural alternatives with the ability to prevent colonization of different biofouling species, from bacteria to macroinvertebrates. A multi-bioassay approach was applied to assess portoamides antifouling properties, marine ecotoxicity and molecular mode of action. Results showed high effectiveness in the prevention of mussel larvae settlement (EC50 = 3.16 µM), and also bioactivity towards growth and biofilm disruption of marine biofouling bacterial strains, while not showing toxicity towards both target and non-target species. Antifouling molecular targets in mussel larvae include energy metabolism modifications (failure in proton-transporting ATPases activity), structural alterations of the gills and protein and gene regulatory mechanisms. Overall, portoamides reveal a broad-spectrum bioactivity towards diverse biofouling species, including a non-toxic and reversible effect towards mussel larvae, showing potential to be incorporated as an active ingredient in antifouling coatings.

Fibre Ultrastructure.

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

The Follicle Cycle in Brief.

This chapter presents a very succinct overview of the cyclic biology of the hair follicle as it transitions from the quiescent telogen stage to the anagen stage in which hairs are actively produced before regressing through the catagen stage to telogen.

Diversity of Trichocyte Keratins and Keratin Associated Proteins.

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.

Development of Hair Fibres.

The growth of hairs occurs during the anagen phase of the follicle cycle. Hair growth begins with basement membrane-bound stem cells (mother cells) around the dermal papilla neck which continuously bud off daughter cells which further divide as a transient amplifying population. Division ceases as cell line differentiation begins, which entails changes in cell junctions, cell shape and position, and cell-line specific cytoplasmic expression of keratin and trichohyalin. As the differentiating cells migrate up the bulb, nuclear function ceases in cortex, cuticle and inner root sheath (IRS) layers. Past the top of the bulb, cell shape/position changes cease, and there is a period of keratin and keratin-associated protein (KAP) synthesis in fibre cell lines, with increases, in particular of KAP species. A gradual keratinization process begins in the cortex at this point and then non-keratin cell components are increasingly broken down. Terminal cornification, or hardening, is associated with water loss and precipitation of keratin. In the upper follicle, the hair, now in its mature form, detaches from the IRS, which is then extracted of material and becomes fragmented to release the fibre. Finally, the sebaceous and sudoriferous (if present) glands coat the fibre in lipid-rich material and the fibre emerges from the skin. This chapter follows the origin of the hair growth in the lower bulb and traces the development of the various cell lines.

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.

Proteomic and peptidomic differences and similarities between four muscle types from New Zealand raised Angus steers.

Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. longissimus thoracis et lumborum, m. psoas major and m. infraspinatus) to test their differences and common features in protein and peptide abundances. The ultimate goal of such a comparison is to match muscle types to products with targeted properties. Protein profiling based on two-dimensional electrophoresis showed that the overall profiles were similar, but, between muscle types, significant (p<0.05) intensity differences were observed in twenty four protein spots. Profiling of endogenous peptides allowed characterisation of 346 peptides. Quantitative analysis showed a clear distinction between the muscle types. Forty-four peptides were identified that showed a statistically significant (p<0.05) and substantial (>2-fold change) difference between at least two muscle types. These analyses demonstrate substantial similarities between these four muscle types, but also clear distinctions in their profiles; specifically a 25% difference between at least two muscles at the peptidomic level, and a 14% difference at the proteomic level.