Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells.

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

Shear-induced unidirectional deposition of bacterial cellulose microfibrils using rising bubble stream cultivation.

In crystalline cellulose I, all glucan chains are ordered from reducing ends to non-reducing ends. Thus, the polarity of individual chains is added forming a large dipole within the crystal. If one can engineer unidirectional alignment (parallel packing) of cellulose crystals, then it might be possible to utilize the material properties originating from polar crystalline structures. However, most post-synthesis manipulation methods reported so far can only achieve the uniaxial alignment with bi-directionality (antiparallel packing). Here, we report a method to induce the parallel packing of bacterial cellulose microfibrils by applying unidirectional shear stress during the synthesis and deposition through the rising bubble stream in a culture medium. Driving force for the alignment is explained with mathematical estimation of the shear stress. Evidences of the parallel alignment of crystalline cellulose Iα domains were obtained using nonlinear optical spectroscopy techniques.

The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

PURPOSE: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. METHODS: Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. RESULTS: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. CONCLUSIONS: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers.

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5-1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis.

Supramolecular networks composed of fibrillins (fibrillin-1 and fibrillin-2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three-dimensional basket-like lattice of FMF intersecting basement membranes at the dermal-epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as "fibrillinopathies." In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin-dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF-associated ligands.

Effective Elimination and Biodegradation of Polycyclic Aromatic Hydrocarbons from Seawater through the Formation of Magnetic Microfibres.

Supramolecular aggregates formed between polycyclic aromatic hydrocarbons and either naphthalene or perylene-derived diimides have been anchored in magnetite magnetic nanoparticles. The high affinity and stability of these aggregates allow them to capture and confine these extremely carcinogenic contaminants in a reduced space. In some cases, the high cohesion of these aggregates leads to the formation of magnetic microfibres of several microns in length, which can be isolated from the solution by the direct action of a magnet. Here we show a practical application of bioremediation aimed at the environmental decontamination of naphthalene, a very profuse contaminant, based on the uptake, sequestration, and acceleration of the biodegradation of the formed supramolecular aggregate, by the direct action of a bacterium of the lineage Roseobacter (biocompatible with nanostructured receptors and very widespread in marine environments) without providing more toxicity to the environment.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture.

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD. Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured. Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

Artificially lignified cell wall catalyzed by peroxidase selectively localized on a network of microfibrils from cultured cells.

MAIN CONCLUSION: An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

Heterogeneity of fibrillin-rich microfibrils extracted from human skin of diverse ethnicity.

The dermal elastic fibre network is the primary effector of skin elasticity, enabling it to extend and recoil many times over the lifetime of the individual. Fibrillin-rich microfibrils (FRMs) constitute integral components of the elastic fibre network, with their distribution showing differential deposition in the papillary dermis across individuals of diverse skin ethnicity. Despite these differential findings in histological presentation, it is not known if skin ethnicity influences FRM ultrastructure. FRMs are evolutionarily highly conserved from jellyfish to man and, regardless of tissue type or species, isolated FRMs have a characteristic 'beads-on-a-string' ultrastructural appearance, with an average inter-bead distance (or periodicity) of 56 nm. Here, skin biopsies were obtained from the photoprotected buttock of healthy volunteers (18-27 years; African: n = 5; European: n = 5), and FRMs were isolated from the superficial papillary dermis and deeper reticular dermis and imaged by atomic force microscopy. In the reticular dermis, there was no significant difference in FRM ultrastructure between European and African participants. In contrast, in the more superficial papillary dermis, inter-bead periodicity was significantly larger for FRMs extracted from European participants than from African participants by 2.20 nm (p < .001). We next assessed whether these differences in FRM ultrastructure were present during early postnatal development by characterizing FRMs from full-thickness neonatal foreskin. Analysis of FRM periodicity identified no significant difference between neonatal cohorts (p = .865). These data suggest that at birth, FRMs are developmentally invariant. However, in adults of diverse skin ethnicity, there is a deviation in ultrastructure for the papillary dermal FRMs that may be acquired during the passage of time from child to adulthood. Understanding the mechanism by which this difference in papillary dermal FRMs arises warrants further study.

The systemic influence of chronic smoking on skin structure and mechanical function.

One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Hierarchical levels of organization of the Brazil nut mesocarp.

Aiming to understand Nature´s strategies that inspire new composite materials, the hierarchical levels of organization of the Brazil nut (Bertholletia excelsa) mesocarp were investigated. Optical microscopy, scanning electron microscopy (SEM), microtomography (MicroCT) and small-angle X-ray scattering (SAXS) were used to deeply describe the cellular and fibrillary levels of organization. The mesocarp is the middle layer of the fruit which has developed several strategies to avoid its opening and protect its seed. Fibers have a different orientation in the three layers of the mesocarp, what reduces the anisotropy of the structure. Sclereids cells with thick cell walls fill the spaces between the fibers resembling a foam-filled structural composite. The mesocarp has several tubular channels and fractured surfaces which may work as sites for crack trapping and increase toughness. The thick and lignified cell wall of sclereids and fibers and the weak interface between cells can promote a longer and tortuous intercellular crack path. Additionally, fibers with high strength and stiffness due to microfibrils oriented along the main cell axis (µ = 0° to 17°) were identified in the innermost layer of the mesocarp. Such an understanding of each hierarchical level can inspire the development of new cellular composites with improved mechanical behavior.

Application of confocal, SHG and atomic force microscopy for characterizing the structure of the most superficial layer of articular cartilage.

The surface of articular cartilage plays a crucial role in attenuating and transmitting mechanical loads in synovial joints to facilitate painless locomotion. Disruption to the surface of articular cartilage causes changes to its frictional properties instigating the deterioration of the tissue. In this study, we physically peeled the most superficial layer, a transparent membrane of 20.0 ± 4.7 µm thick, from the central loading region of femoral condyles of sheep. The ultrastructure of this layer without interference from the underlying cartilage was independently investigated using confocal, second harmonic generation and atomic force microscopy. We found that the most superficial layer contains chondrocytes, densely packed collagen, coarse elastic fibres and a fine elastic network. The elastic fibres are most prevalent at the surface of the layer, where collagen and chondrocyte densities are lowest. At the interface of this most superficial layer with the underlying bulk cartilage, a dense fibrillar network exists, formed mainly by collagen fibrils and elastin microfibrils. By contrast, the interface of the underlying cartilage with the most superficial layer contains collagen fibrils, fine microfibrils and microfibrils distinctively laced on one side. The findings of this study will play an important role in understanding the mechanical function and wear resistance of articular cartilage, and in developing more promising tissue engineering techniques to treat cartilage defects and osteoarthritis. LAY DESCRIPTION: The chronic pain and dysfuction in synovial joints caused by osteoarthritis can have a debilitating impact on daily activities for sufferers. Osteoarthritis is characterised by the deterioration of the articular cartilage. Despite intensive research, the wear mechanism of articular cartilage and the progression of osteoarthritis remain unclear in the literature. Articular cartilage is a resilient tissue that provides a low friction surface to facilitate painless locomotion. The surface of articular cartilage plays a crucial role in attenuating and transmitting mechanical loads. Disruption at the surface of articular cartilage causes changes to its frictional properties, instigating the deterioration of the tissue. Despite this, the definition of the most superficial layer of articular cartilage, as well as its composition and microstructure, have endured a long history of debate, clouding our understanding of the early progression of osteoarthritis. In order to investigate the surface of articular cartilage independently from the underlying cartilage, we physically peeled a transparent membrane of 20.0 ± 4.7 µm thickness, the most superficial layer, from the central loading region of the femoral condyles of sheep. Using confocal, second harmonic generation and atomic force microscopy, we found that the most superficial layer contains cartilage cells (chondrocytes), densely packed collagen, coarse elastic fibres and a fine elastic network. The coarse elastic fibres are most prevalent at the surface of the layer where collagen and chondrocyte densities are lowest. Furthermore, we investigated the surfaces at the interface of the most superficial layer with the underlying articular cartilage. At the interface of this most superficial layer with the underlying bulk cartilage, a dense fibrillar network exists, formed mainly by collagen fibrils and elastin microfibrils. In contrast, the interface of the underlying cartilage with the most superficial layer contains collagen fibrils, fine microfibrils and microfibrils distinctively laced on one side. The findings of this study have confirmed that there is a most superficial layer that is able to be removed using a tangential force. Through the application of advanced imaging technologies, we have shown that this most superficial layer is cellular and have detailed its composition and ultrastructure. Due to the close association between the form and function of tissues, the findings of this study will play an important role in understanding the mechanical function and wear mechanism of articular cartilage. This may lead to the development of more promising tissue engineering techniques to treat cartilage defects and osteoarthritis.

Developmental and age-related changes to the elastic lamina of Bruch's membrane in mice.

BACKGROUND: Fibrillin-1, tropoelastin, fibulin-5, and latent transforming growth factor beta-binding protein-2 and protein-4 (LTBP-2 and LTBP-4) are essential proteins for the elastic lamina (EL). In this study, we analyzed each of these molecules in the EL of Bruch's membrane (BM) through development and aging. METHODS: C57BL/6 mice (embryonic (E) days E12.5, E15.5, and E18.5; postnatal (P) days P1, P4, and P7 and P3, P6, and P75 weeks of age) were used. To investigate localization, immunohistochemical staining (IH) was performed. Transmission electron microscopy (TEM) was used to evaluate the formation of microfibrils and tropoelastin. mRNA expression was determined by quantitative real-time PCR (qRT-PCR). RESULTS: All five proteins were expressed in the EL of BM by IH except in embryonic mice. TEM results showed that tropoelastin co-stained with microfibrils. Between 3 and 6 weeks of age, microfibrils became longer and thicker. It was difficult to evaluate the EL of BM in senile mice at 75 weeks of age because of abundant deposits which correspond to drusen. mRNA levels of each protein increased dramatically from E15.5 to P1 days and plateaued by P3 weeks as shown by qRT-PCR. CONCLUSIONS: In conclusion, these five proteins are possibly involved in elastic fiber assembly in BM. We define the date of full assembly of the EL of BM as 3 weeks of age in mice.

Resonant soft X-ray scattering reveals cellulose microfibril spacing in plant primary cell walls.

Cellulose microfibrils are crucial for many of the remarkable mechanical properties of primary cell walls. Nevertheless, many structural features of cellulose microfibril organization in cell walls are not yet fully described. Microscopy techniques provide direct visualization of cell wall organization, and quantification of some aspects of wall microstructure is possible through image processing. Complementary to microscopy techniques, scattering yields structural information in reciprocal space over large sample areas. Using the onion epidermal wall as a model system, we introduce resonant soft X-ray scattering (RSoXS) to directly quantify the average interfibril spacing. Tuning the X-ray energy to the calcium L-edge enhances the contrast between cellulose and pectin due to the localization of calcium ions to homogalacturonan in the pectin matrix. As a consequence, RSoXS profiles reveal an average center-to-center distance between cellulose microfibrils or microfibril bundles of about 20 nm.

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils.

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.

Structural and compositional diversity of fibrillin microfibrils in human tissues.

Elastic fibers comprising fibrillin microfibrils and elastin are present in many tissues, including the skin, lungs, and arteries, where they confer elasticity and resilience. Although fibrillin microfibrils play distinct and tissue-specific functional roles, it is unclear whether their ultrastructure and composition differ between elastin-rich (skin) and elastin-poor (ciliary body and zonule) organs or after in vitro synthesis by cultured cells. Here, we used atomic force microscopy, which revealed that the bead morphology of fibrillin microfibrils isolated from the human eye differs from those isolated from the skin. Using newly developed pre-MS preparation methods and LC-MS/MS, we detected tissue-specific regions of the fibrillin-1 primary structure that were differentially susceptible to proteolytic extraction. Comparing tissue- and culture-derived microfibrils, we found that dermis- and dermal fibroblast-derived fibrillin microfibrils differ in both bead morphology and periodicity and also exhibit regional differences in fibrillin-1 proteolytic susceptibility. In contrast, collagen VI microfibrils from the same dermal or fibroblast samples were invariant in ultrastructure (periodicity) and protease susceptibility. Finally, we observed that skin- and eye-derived microfibril suspensions were enriched in elastic fiber- and basement membrane-associated proteins, respectively. LC-MS/MS also identified proteins (such as calreticulin and protein-disulfide isomerase) that are potentially fundamental to fibrillin microfibril biology, regardless of their tissue source. Fibrillin microfibrils synthesized in cell culture lacked some of these key proteins (MFAP2 and -4 and fibrillin-2). These results showcase the structural diversity of these key extracellular matrix assemblies, which may relate to their distinct roles in the tissues where they reside.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Electrospun Phospholipid Fibers as Micro-Encapsulation and Antioxidant Matrices.

Electrospun phospholipid (asolectin) microfibers were investigated as antioxidants and encapsulation matrices for curcumin and vanillin. These phospholipid microfibers exhibited antioxidant properties which increased after the encapsulation of both curcumin and vanillin. The total antioxidant capacity (TAC) and the total phenolic content (TPC) of curcumin/phospholipid and vanillin/phospholipid microfibers remained stable over time at different temperatures (refrigerated, ambient) and pressures (vacuum, ambient). ¹H-NMR confirmed the chemical stability of both encapsulated curcumin and vanillin within phospholipid fibers. Release studies in aqueous media revealed that the phenolic bioactives were released mainly due to swelling of the phospholipid fiber matrix over time. The above studies confirm the efficacy of electrospun phospholipid microfibers as encapsulation and antioxidant systems.

High-Resolution Field Emission Scanning Electron Microscopy (FESEM) Imaging of Cellulose Microfibril Organization in Plant Primary Cell Walls.

We have used field emission scanning electron microscopy (FESEM) to study the high-resolution organization of cellulose microfibrils in onion epidermal cell walls. We frequently found that conventional "rule of thumb" conditions for imaging of biological samples did not yield high-resolution images of cellulose organization and often resulted in artifacts or distortions of cell wall structure. Here we detail our method of one-step fixation and dehydration with 100% ethanol, followed by critical point drying, ultrathin iridium (Ir) sputter coating (3 s), and FESEM imaging at a moderate accelerating voltage (10 kV) with an In-lens detector. We compare results obtained with our improved protocol with images obtained with samples processed by conventional aldehyde fixation, graded dehydration, sputter coating with Au, Au/Pd, or carbon, and low-voltage FESEM imaging. The results demonstrated that our protocol is simpler, causes little artifact, and is more suitable for high-resolution imaging of cell wall cellulose microfibrils whereas such imaging is very challenging by conventional methods.

A new in vitro assay to test UVR protection of dermal extracellular matrix components by a flat spectrum sunscreen.

The efficacy of topical sunscreens is currently assessed by crude, costly and time consuming in vivo assays. We have previously demonstrated that components of the dermal extracellular matrix (ECM), rich in UV-absorbing amino acids, are susceptible to damage by solar simulated radiation (SSR) in vitro. Here we developed an in vitro method to test the ability of sunscreens to protect fibrillin-rich microfibrils (FRM) and fibronectin, key components of the dermal ECM from UV-induced damage. Solutions of FRM or fibronectin were irradiated without protection, in the presence of a vehicle or a commercially-available flat-spectrum sunscreen. The effect of SSR on molecular structure was determined by atomic force microscopy (FRM) and SDS-PAGE (fibronectin). Following irradiation, FRM periodicity became bi-modally distributed (peaks: 40nm & 59nm) compared to the unimodal distribution in unexposed controls (peak: 50nm). Irradiation in the presence of flat-spectrum sunscreen protected against this change, maintaining the unimodal distribution. SSR induced significant aggregation of fibronectin (p=0.005), which was abrogated by sunscreen. These results demonstrate that this in vitro assay system is sufficiently sensitive to act as an initial/additional screen of sunscreen efficacy. We conclude that sunscreen can reduce UV-mediated damage of key dermal ECM in vitro and thereby prevent remodelling associated with photoageing.