Investigating the origins of nanostructural variations in differential ethnic hair types using X-ray scattering techniques.

OBJECTIVE: Human hair is a major determinant of visual ethnic differentiation. Although hair types are celebrated as part of our ethnic diversity, the approach to hair care has made the assumption that hair types are structurally and chemically similar. Although this is clearly not the case at the macroscopic level, the intervention of many hair treatments is at the nanoscopic and molecular levels. The purpose of the work presented here is to identify the main nanoscopic and molecular hierarchical differences across five different ethnic hair types from hair fibres taken exclusively from the scalp. These are Afro (subdivided into elastic 'rubber' and softer non-elastic 'soft'), Chinese, European and Mullato (mixed race). METHODS: Small angle X-Ray scattering (SAXS) is a technique capable of resolving nanostructural variations in complex materials. Individual hair fibres from different ethnic hair types were used to investigate structural features found in common and also specific to each type. Simultaneous wide angle X-Ray scattering (WAXS) was used to analyse the submolecular level structure of the fibrous keratin present. The data sets from both techniques were analysed with principal component analysis (PCA) to identify underlying variables. RESULTS: Principal component analysis of both SAXS and WAXS data was shown to discriminate the scattering signal between different hair types. The X-ray scattering results show a common underlying keratin intermediate filament (KIF) structure. However, distinct differences were observed in the preferential orientation and intensity signal from the lipid component of the hair. In addition, differences were observed in the intensity distribution of the very low-angle sample-dependent diffuse scatter surrounding the 'beamstop.' CONCLUSION: The results indicate that the fibrous keratin scaffold remains consistent between ethnic hair types. The hierarchies made by these may be modulated by variation in the content of keratin-associated proteins (KAPs) and lipids that alter the interfacial structures and lead to macroscopic differences in hair morphology.

Bimodal collagen fibril diameter distributions direct age-related variations in tendon resilience and resistance to rupture.

Scaling relationships have been formulated to investigate the influence of collagen fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (D(D1)) and larger (D(D2)) mean Ds, respectively. Both D(D1) and D(D2) increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) u(E) [from initial loading until the yield stress (σ(Y))], which contributes primarily to tendon resilience, and 2) u(F) [from σ(Y) through the maximum stress (σ(U)) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities u(E)/σ(Y) and u(F)/σ(U), both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in u(E)/σ(Y) (resilience energy) are associated with decreases in D(D1) and increases in D(D2), whereas u(F)/σ(U) (rupture energy) is associated with increases in D(D1) alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.

Protein and mineral characterisation of rendered meat and bone meal.

We report the characterisation of meat and bone meal (MBM) standards (Set B-EFPRA) derived from cattle, sheep, pig and chicken, each rendered at four different temperatures (133, 137, 141 and 145 °C). The standards, prepared for an EU programme STRATFEED (to develop new methodologies for the detection and quantification of illegal addition of mammalian tissues in feeding stuffs), have been widely circulated and used to assess a range of methods for identification of the species composition of MBM. The overall state of mineral alteration and protein preservation as a function of temperature was monitored using small angle X-ray diffraction (SAXS), amino acid composition and racemization analyses. Progressive increases in protein damage and mineral alteration in chicken and cattle standards was observed. In the case of sheep and pig, there was greater damage to the proteins and alteration of the minerals at the lowest treatment temperature (133 °C), suggesting that the thermal treatments must have been compromised in some way. This problem has probably impacted upon the numerous studies which tested methods against these heat treatments. We use protein mass spectrometric methods to explore if thermostable proteins could be used to identify rendered MBM. In more thermally altered samples, so-called 'thermostable' proteins such as osteocalcin which has been proposed as a ideal target to speciate MBM were no longer detectable, but the structural protein type I collagen could be used to differentiate all four species, even in the most thermally altered samples.

Effects of frozen storage temperature on the elasticity of tendons from a small murine model.

The basic mechanism of reinforcement in tendons addresses the transfer of stress, generated by the deforming proteoglycan (PG)-rich matrix, to the collagen fibrils. Regulating this mechanism involves the interactions of PGs on the fibril with those in the surrounding matrix and between PGs on adjacent fibrils. This understanding is key to establishing new insights on the biomechanics of tendon in various research domains. However, the experimental designs in many studies often involved long sample preparation time. To minimise biological degradation the tendons are usually stored by freezing. Here, we have investigated the effects of commonly used frozen storage temperatures on the mechanical properties of tendons from the tail of a murine model (C57BL6 mouse). Fresh (unfrozen) and thawed samples, frozen at temperatures of -20°C and -80°C, respectively, were stretched to rupture. Freezing at -20°C revealed no effect on the maximum stress (σ), stiffness (E), the corresponding strain (ε) at σ and strain energy densities up to ε (u) and from ε until complete rupture (up). On the other hand, freezing at -80°C led to higher σ, E and u; ε and up were unaffected. The results implicate changes in the long-range order of radially packed collagen molecules in fibrils, resulting in fibril rupture at higher stresses, and changes to the composition of extrafibrillar matrix, resulting in an increase in the interaction energy between fibrils via collagen-bound PGs.

Ageing changes in the tensile properties of tendons: influence of collagen fibril volume fraction.

Connective tissues are biological composites comprising of collagen fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular matrices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, fibril diameter. However, quantitative attempts to correlate fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength (sigma) and stiffness (E) on the collagen fibril cross-sectional area fraction (rho), which is related to the fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6-35.3 months old were stretched to failure to determine sigma and E. Parallel measurements of rho as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of fibrils reinforcing a PG gel in tendons were used to investigate the influence of rho on ageing changes in sigma and E. The magnitudes of sigma, E, and rho increased rapidly from 1.6 months to 4.0 months (P-values <0.05) before reaching a constant (age independent) from 4.0 months to 29.0 months (P-values >0.05); this trend continued for E and rho (P-values >0.05) from 29.0 months to 35.3 months, but not for sigma, which decreased gradually (P-values <0.05). Linear regression analysis revealed that age-related changes in sigma and E correlated positively to rho (P-values <0.05). Collagen fibril cross-sectional area fraction rho is a significant predictor of ageing changes in sigma and E in the tail tendons of C57BL6 mice.

The variability in type I collagen helical pitch is reflected in the D periodic fibrillar structure.

The variability in amino acid axial rise per residue of the collagen helix is a potentially important parameter that is missing in many structural models of fibrillar collagen to date. The significance of this variability has been supported by evidence from collagen axial structures determined by electron microscopy and X-ray diffraction, as well as studies of the local sequence-dependent conformation of the collagen helix. Here, sequence-dependent variation of the axial rise per residue was used to improve the fit between simulated diffraction patterns derived from model structures of the axially projected microfibrillar structure and the observed X-ray diffraction pattern from hydrated rat tail tendon. Structural models were adjusted using a genetic algorithm that allowed a wide range of structures to be tested efficiently. The results show that variation of the axial rise per residue could reduce the difference metric between model and observed data by up to 50%, indicating that such a variable is a necessary part of fibril model structure building. The variation in amino acid translation was also found to be influenced by the number of proline and hydroxyproline residues in the triple helix structure.

Domains 17-27 of tropoelastin contain key regions of contact for coacervation and contain an unusual turn-containing crosslinking domain.

The central region of tropoelastin including domains 19-25 of human tropoelastin forms a hot-spot for contacts during the inter-molecular association of tropoelastin by coacervation [Wise, S.G., Mithieux, S.M., Raftery, M.J. and Weiss, A.S (2005). "Specificity in the coacervation of tropoelastin: solvent exposed lysines." Journal of Structural Biology 149: 273-81.]. We explored the physical properties of this central region using a sub-fragment bordered by domains 17-27 of human tropoelastin (SHEL 17-27) and identified the intra- and inter-molecular contacts it forms during coacervation. A homobifunctional amine reactive crosslinker (with a maximum reach of 11 A, corresponding to approximately 7 residues in an extended polypeptide chain) was used to capture these contacts and crosslinked regions were identified after protease cleavage and mass spectrometry (MS) with MS/MS verification. An intermolecular crosslink formed between the lysines at positions 353 of each strand of tropoelastin at the lowest of crosslinker concentrations and was observed in all samples tested, suggesting that this residue forms an important initial contact during coacervation. At higher crosslinker concentrations, residues K425 and K437 showed the highest levels of involvement in crosslinks. An intramolecular crosslink between these K425 and K437, separated by 11 residues, indicated that a structural bend must serve to bring these residues into close proximity. These studies were complemented by small angle X-ray scattering studies that confirmed a bend in this important subfragment of the tropoelastin molecule.

Nanoarchitectures of the animal extracellular matrix: opportunities for synchrotron radiation studies on collagen and fibrillin.

The extracellular matrix comprises structures that support the architectural organization of virtually all animal tissues. Within this architecture, two classes of protein assemblies found as long slender fibrils (collagen and fibrillin) characterize the bulk of the extracellular matrix. In both classes of fibrous protein, the molecular organization within a fibril ensures that the properties of the individual molecules transcend to the nanostructural and mesoscopic levels of structural organization and thence the tissue itself. The composition of the fibrils, in conjunction with other biomolecules and their suprafibrillar architecture, facilitates the formation of tissues as diverse as skin, tendon, cornea ciliary zonules and aorta. Here the relative tear resistance, strength, transparency and optical properties are paramount for proper function. Many structural investigations of fibrous protein structure have relied heavily on the use of synchrotron radiation in order to elucidate molecular packing, primarily due to the distinct benefits that X-ray diffraction provides, such as minimal sample preparation, rapid data collection and in situ mechanical testing. In this paper, an overview of the investigations that have revealed different levels of molecular architecture in fibril-based tissues is presented. Emerging future technology and how this can be matched with the pressing questions in extracellular matrix biology are also discussed.

A preliminary study of breast cancer diagnosis using laboratory based small angle x-ray scattering.

Breast tissue collected from tumour samples and normal tissue from bi-lateral mastectomy procedures were examined using small angle x-ray scattering. Previous work has indicated that breast tissue disease diagnosis could be performed using small angle x-ray scattering (SAXS) from a synchrotron radiation source. The technique would be more useful to health services if it could be made to work using a conventional x-ray source. Consistent and reliable differences in x-ray scatter distributions were observed between samples from normal and tumour tissue samples using the laboratory based 'SAXSess' system. Albeit from a small number of samples, a sensitivity of 100% was obtained. This result encourages us to pursue the implementation of SAXS as a laboratory based diagnosis technique.

Evidence of a distinct lipid fraction in historical parchments: a potential role in degradation?

Parchment, a biologically based material obtained from the processed hides of animals such as cattle and sheep, has been used for millennia as a writing medium. Although numerous studies have concentrated on the structure and degradation of collagen within parchment, little attention has been paid to noncollagenous components, such as lipids. In this study, we present the results of biochemical and structural analyses of historical and newly manufactured parchment to examine the potential role that lipid plays in parchment stability. The lipid fraction extracted from the parchments displayed different fatty acid compositions between historical and reference materials. Gas chromatography, small-angle X-ray scattering, and solid-state NMR were used to identify and investigate the lipid fraction from parchment samples and to study its contribution to collagen structure and degradation. We hypothesize that the origin of this lipid fraction is either intrinsic, attributable to incomplete fat removal in the manufacturing process, or extrinsic, attributable to microbiological attack on the proteinaceous component of parchments. Furthermore, we consider that the possible formation of protein-lipid complexes in parchment over the course of oxidative degradation may be mediated by reactive oxygen species formed by lipid peroxidation.

Collagen fibril form and function.

The majority of collagen in the extracellular matrix is found in a fibrillar form, with long slender filaments each displaying a characteristic approximately 67?nm D-repeat. Here they provide the stiff resilient part of many tissues, where the inherent strength of the collagen triple helix is translated through a number of hierarchical levels to endow that tissue with its specific mechanical properties. A number of collagen types have important structural roles, either comprising the core of the fibril or decorating the fibril surface to give enhanced functionality. The architecture of subfibrillar and suprafibrillar structures (such as microfibrils), lateral crystalline and liquid crystal ordering, interfibrillar interactions, and fibril bundles is described. The fibril surface is recognized as an area that contains a number of intimate interactions between different collagen types and other molecular species, especially the proteoglycans. The interplay between molecular forms at the fibril surface is discussed in terms of their contribution to the regulation of fibril diameter and their role in interfibrillar interactions.

Molecular interactions in collagen and chitosan blends.

Molecular interactions between collagen and chitosan (CC) have the potential to produce biocomposites with novel properties. We have characterised the molecular interactions in CC complexes by viscometry, wide angle X-ray scattering and Fourier transform infrared spectroscopy. It was found that CC are miscible at the molecular level and exhibit interactions between the components; X-ray diffraction of CC blends indicate that the collagen helix structure is lost in CC films with increasing chitosan content. Non-linear viscometic behaviour with decreasing chitosan content is interpreted as evidence of a third structural phase formed as a complex of CC. The blending of collagen with chitosan gives the possibility of producing new bespoke materials for potential biomedical applications.

Bone mineral change during experimental heating: an X-ray scattering investigation.

The effects of heating and burning on bone mineral have previously been studied using techniques such as X-ray diffraction (XRD) with the aim of discerning a characteristic signature of crystal change. This would enable a better understanding of alteration to bone mineral during heating, which would in turn impact on the preparation and use of natural bone hydroxyapatite as a biomaterial resource. In addition, this knowledge could prove invaluable in the investigation of burned human remains from forensic and archaeological contexts in cremation and funerary practice. Here we describe a complementary method, small-angle X-ray scattering (SAXS), to determine more accurately the changes to bone crystallite size and shape during an experimental heating regimen. Samples were subjected to controlled heating at 500 degrees C, 700 degrees C, or 900 degrees C for 15 or 45 min. Our results show bone crystallites begin to alter in the first 15 min of heating to 500 degrees C or above. They then appear to stabilise to a temperature-specific thickness and shape with prolonged heating. While the samples heated to lower temperatures or for shorter periods produce XRD traces showing little alteration to the apatite, corresponding information obtained from SAXS shows an early, subtle change in crystal parameters.

Young's modulus varies with differential orientation of keratin in feathers.

Feathers are composed of a structure that, whilst being very light, is able to withstand the large aerodynamic forces exerted upon them during flight. To explore the contribution of molecular orientation to feather keratin mechanical properties, we have examined the nanoscopic organisation of the keratin molecules by X-ray diffraction techniques and have confirmed a link between this and the Young's modulus of the feather rachis. Our results indicate that along the rachis length, from calamus to tip, the keratin molecules become more aligned than at the calamus before returning to a state of higher mis-orientation towards the tip of the rachis. We have also confirmed the general trend of increasing Young's modulus with distance along the rachis. Furthermore, we report a distinct difference in the patterns of orientation of beta-keratin in the feathers of flying and flightless birds. The trend for increased modulus along the feathers of volant birds is absent in the flightless ostrich.

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study.

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.

Microfocus small angle X-ray scattering reveals structural features in archaeological bone samples: detection of changes in bone mineral habit and size.

Microfocus X-ray scattering provides a powerful nondestructive technique capable of providing important information about the size, habit, and arrangement of mineral crystals in bone. The technique is capable of probing textural differences in a sample at a micron scale resolution. The study presented here involved the analysis of a number of archaeological bones by microfocus X-ray scattering at the ESRF Grenoble in order to determine local changes in mineral durability. The results showed that regions of bone with a modified microscopic morphology contained a greater dispersion of crystal shape when compared with more intact regions and control contemporary bone samples, but the crystal thickness values showed similar consistency. We speculate that the persistence of collagen in the archaeological bone may allow diagenetic remodeling of bone in terms of crystallite shape but defines the size of remodelled crystallites. The ability to detect such local changes in texture has wide potential for determining crystal characteristics in healthy and diseased bone samples.

Fibrillin-rich microfibrils: elastic biopolymers of the extracellular matrix.

Fibrillin-rich microfibrils are evolutionarily ancient macromolecular assemblies of the extracellular matrix. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. Microfibril extensibility supports the low pressure closed circulations of lower organisms such as crustaceans. In higher vertebrates, microfibrils act as a template for elastin deposition and are components of mature elastic fibres. In man, the importance of microfibrils is highlighted by the linkage of mutations in their principal structural component, fibrillin-1, to the heritable disease Marfan syndrome which is characterised by severe cardiovascular, skeletal and ocular defects. When isolated from tissues, fibrillin-rich microfibrils have a complex ultrastructural organisation with a characteristic 'beads-on-a-strong' appearance. X-ray fibre diffraction studies and biomechanical testing have shown that microfibrils are reversibly extensible at tissue extensions of 100%. Ultrastructural analysis and 3D reconstructions of isolated microfibrils using automated electron tomography have revealed new details of how fibrillin molecules are aligned within microfibrils in untensioned and extended states, and delineated the role of calcium in regulating microfibril beaded periodicity, rest length and molecular organisation. The molecular basis of how fibrillin molecules assemble into microfibrils, the central role of cells in regulating this process, and the identity of other molecules that may coassemble into microfibrils are now being elucidated. This information will enhance our understanding of the elastic mechanism of these unique extracellular matrix polymers, and may lead to new microfibril-based strategies for repairing elastic tissues in ageing and disease.

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.

Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.

An analysis of the lamellar structure of sea urchin egg cortical granules using X-ray scattering.

Cortical granules (CGs) are secretory vesicles associated with egg and oocyte plasma membranes that undergo exocytosis at fertilisation. In the sea urchin Strongylocentrotus purpuratus, the internal organisation of these CGs exhibits a lamellar-type morphology. The different lamellar layers correspond to proteoglycans, structural proteins and enzymes required for fertilisation envelope assembly and modification of the post-fertilisation egg surface. We have studied the lamellar structure of CGs using X-ray scattering and reveal the contrast density variation of the lamellae in the native state. The structure of functionally competent CGs in situ differs significantly from that determined by electron microscopic studies. We observed a strong periodicity of the lamellar structure of 280 A as opposed to the 590 A repeat observed previously. Fusion of the CGs produced a loss of the lamellar repeat and the development of a broad peak corresponding to a 20 A periodicity that may be indicative of the molecular packing in the resulting hydrated gel structure.