Invasive mussels fashion silk-like byssus via mechanical processing of massive horizontally acquired coiled coils.

Zebra and quagga mussels (Dreissena spp.) are invasive freshwater biofoulers that perpetrate devastating economic and ecological impact. Their success depends on their ability to anchor onto substrates with protein-based fibers known as byssal threads. Yet, compared to other mussel lineages, little is understood about the proteins comprising their fibers or their evolutionary history. Here, we investigated the hierarchical protein structure of Dreissenid byssal threads and the process by which they are fabricated. Unique among bivalves, we found that threads possess a predominantly ß-sheet crystalline structure reminiscent of spider silk. Further analysis revealed unexpectedly that the Dreissenid thread protein precursors are mechanoresponsive α-helical proteins that are mechanically processed into ß-crystallites during thread formation. Proteomic analysis of the byssus secretory organ and byssus fibers revealed a family of ultrahigh molecular weight (354 to 467 kDa) asparagine-rich (19 to 20%) protein precursors predicted to form α-helical coiled coils. Moreover, several independent lines of evidence indicate that the ancestral predecessor of these proteins was likely acquired via horizontal gene transfer. This chance evolutionary event that transpired at least 12 Mya has endowed Dreissenids with a distinctive and effective fiber formation mechanism, contributing significantly to their success as invasive species and possibly, inspiring new materials design.

Advanced materials design based on waste wood and bark.

Trees belong to the largest living organisms on Earth and plants in general are one of our main renewable resources. Wood as a material has been used since the beginning of humankind. Today, forestry still provides raw materials for a variety of applications, for example in the building industry, in paper manufacturing and for various wood products. However, many parts of the tree, such as reaction wood, branches and bark are often discarded as forestry residues and waste wood, used as additives in composite materials or burned for energy production. More advanced uses of bark include the extraction of chemical substances for glues, food additives or healthcare, as well as the transformation to advanced carbon materials. Here, we argue that a proper understanding of the internal fibrous structure and the resulting mechanical behaviour of these forest residues allows for the design of materials with greatly varying properties and applications. We show that simple and cheap treatments can give tree bark a leather-like appearance that can be used for the construction of shelters and even the fabrication of woven textiles. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Unraveling the Rapid Assembly Process of Stiff Cellulosic Fibers from Mistletoe Berries.

The mucilaginous viscin tissue within mistletoe berries possesses an extraordinary ability to be rapidly processed under ambient conditions into stiff cellulosic fibers (>14 GPa) through simple mechanical drawing. This rapid and extreme transformation process is hydration-dependent and involves an astonishing >200-fold increase in length, providing a relevant role model for efforts to produce advanced composites from cellulose-based structures such as cellulose nanocrystals or cellulose nanofibrils. Using a combination of in situ polarized light microscopy, synchrotron X-ray scattering, and humidity-controlled mechanical analysis, we examine here the dynamic transition of a viscin cell bundle from hydrogel-like tissues to high-performance fibers. Our findings indicate a massive phase transition in which cellulose microfibrils containing high-aspect-ratio crystalline domains undergo dramatic reorganization, facilitated by a water-responsive noncellulosic matrix. Transition from an aligned, yet flowing state to a stiff fiber is likely triggered by rapid water loss below 45% relative humidity. These findings not only help understanding the adaptive success of mistletoe but may also be relevant for the development of new facile processing methods for next-generation cellulosic composites.

Self-healing silk from the sea: role of helical hierarchical structure in Pinna nobilis byssus mechanics.

The byssus fibers of Mytilus mussel species have become an important role model in bioinspired materials research due to their impressive properties (e.g. high toughness, self-healing); however, Mytilids represent only a small subset of all byssus-producing bivalves. Recent studies have revealed that byssus from other species possess completely different protein composition and hierarchical structure. In this regard, Pinna nobilis byssus is especially interesting due to its very different morphology, function and its historical use for weaving lightweight golden fabrics, known as sea silk. P. nobilis byssus was recently discovered to be comprised of globular proteins organized into a helical protein superstructure. In this work, we investigate the relationships between this hierarchical structure and the mechanical properties of P. nobilis byssus threads, including energy dissipation and self-healing capacity. To achieve this, we performed in-depth mechanical characterization, as well as tensile testing coupled with in situ X-ray scattering. Our findings reveal that P. nobilis byssus, like Mytilus, possesses self-healing and energy damping behavior and that the initial elastic behavior of P. nobilis byssus is due to stretching and unraveling of the previously observed helical building blocks comprising the byssus. These findings have biological relevance for understanding the convergent evolution of mussel byssus for different species, and also for the field of bio-inspired materials.

A new twist on sea silk: the peculiar protein ultrastructure of fan shell and pearl oyster byssus.

Numerous mussel species produce byssal threads - tough proteinaceous fibers, which anchor mussels in aquatic habitats. Byssal threads from Mytilus species, which are comprised of modified collagen proteins - have become a veritable archetype for bio-inspired polymers due to their self-healing properties. However, threads from different species are comparatively much less understood. In particular, the byssus of Pinna nobilis comprises thousands of fine fibers utilized by humans for millennia to fashion lightweight golden fabrics known as sea silk. P. nobilis is very different from Mytilus from an ecological, morphological and evolutionary point of view and it stands to reason that the structure-function relationships of its byssus are distinct. Here, we performed compositional analysis, X-ray diffraction (XRD) and transmission electron microscopy (TEM) to investigate byssal threads of P. nobilis, as well as a closely related bivalve species (Atrina pectinata) and a distantly related one (Pinctada fucata). This comparative investigation revealed that all three threads share a similar molecular superstructure comprised of globular proteins organized helically into nanofibrils, which is completely distinct from the Mytilus thread ultrastructure, and more akin to the supramolecular organization of bacterial pili and F-actin. This unexpected discovery hints at a possible divergence in byssus evolution in Pinnidae mussels, perhaps related to selective pressures in their respective ecological niches.

Adhesives free bark panels: An alternative application for a waste material.

The proportion of bark in tree trunks is in the range of ~ 10-20%. This large amount of material is currently mainly considered as a by- or even waste-product by the timber processing industry. Recently, efforts towards the use of bark have been made, e.g. as a raw material to harvest different chemical compounds or as an additive for wood particle boards. Our motivation for this work was to keep the bark in an almost natural state and explore alternative processes and applications for use. The traditional method of de-barking tree trunks by peeling was used to harvest large bark pieces. Two pieces of peeled bark were placed crosswise, with the rhytidom side (outer bark) facing each other. After different conditioning steps, bark pieces were hot pressed to panels without adding adhesives. These experiments on bark samples of different Central European tree species suggest that production of panels with species dependent properties is possible and feasible. This is a step towards producing sustainable panels by using a natural waste material, while retaining its beneficial structure and its natural chemical composition.

Mistletoe viscin: a hygro- and mechano-responsive cellulose-based adhesive for diverse material applications.

Mistletoe viscin is a natural cellulosic adhesive consisting of hierarchically organized cellulose microfibrils (CMFs) surrounded by a humidity-responsive matrix that enables mechanical drawing into stiff and sticky fibers. Here, we explored the processability and adhesive capacity of viscin and demonstrated its potential as a source material for various material applications, as well as a source for bioinspired design. Specifically, we revealed that viscin fibers exhibit humidity-activated self-adhesive properties that enable "contact welding" into complex 2D and 3D architectures under ambient conditions. We additionally discovered that viscin can be processed into stiff and transparent free-standing films via biaxial stretching in the hydrated state, followed by drying, whereby CMFs align along local stress fields. Furthermore, we determined that viscin adheres strongly to both synthetic materials (metals, plastics, and glass) and biological tissues, such as skin and cartilage. In particular, skin adhesion makes viscin a compelling candidate as a wound sealant, as we further demonstrate. These findings highlight the enormous potential of this hygro- and mechano-responsive fiber-reinforced adhesive for bioinspired and biomedical applications.

Mineralization generates megapascal contractile stresses in collagen fibrils.

During bone formation, collagen fibrils mineralize with carbonated hydroxyapatite, leading to a hybrid material with excellent properties. Other minerals are also known to nucleate within collagen in vitro. For a series of strontium- and calcium-based minerals, we observed that their precipitation leads to a contraction of collagen fibrils, reaching stresses as large as several megapascals. The magnitude of the stress depends on the type and amount of mineral. Using in-operando synchrotron x-ray scattering, we analyzed the kinetics of mineral deposition. Whereas no contraction occurs when the mineral deposits outside fibrils only, intrafibrillar mineralization generates fibril contraction. This chemomechanical effect occurs with collagen fully immersed in water and generates a mineral-collagen composite with tensile fibers, reminiscent of the principle of reinforced concrete.

Cellulose lattice strains and stress transfer in native and delignified wood.

Small specimens of spruce wood with different degrees of delignification were studied using in-situ tensile tests and simultaneous synchrotron X-ray diffraction to reveal the effect of delignification and densification on their tensile properties at relative humidities of 70-80 %. In addition to mechanical properties, these analyses yield the ratio of strains in the cellulose crystals and in the bulk, which reflects the stress-transfer to crystalline cellulose. While the specific modulus of elasticity slightly increases from native wood by partial or complete delignification, the lattice strain ratio does not show a significant change. This could indicate a compensatory effect from the decomposition of the amorphous matrix by delignification and from a tighter packing of cellulose crystals that would increase the stress transfer. The reduced strain to failure and maximum lattice strain of delignified specimens suggests that the removal of lignin affects the stress-strain behavior with fracture at lower strain levels.

Structure and composition of the tunic in the sea pineapple Halocynthia roretzi: A complex cellulosic composite biomaterial.

Biological organisms produce high-performance composite materials, such as bone, wood and insect cuticle, which provide inspiration for the design of novel materials. Ascidians (sea squirts) produce an organic exoskeleton, known as a tunic, which has been studied quite extensively in several species. However, currently, there are still gaps in our knowledge about the detailed structure and composition of this cellulosic biocomposite. Here, we investigate the composition and hierarchical structure of the tough tunic from the species Halocynthia roretzi, through a cross-disciplinary approach combining traditional histology, immunohistochemistry, vibrational spectroscopy, X-ray diffraction, and atomic force and electron microscopies. The picture emerging is that the tunic of H. roretzi is a hierarchically-structured composite of cellulose and proteins with several compositionally and structurally distinct zones. At the surface is a thin sclerotized cuticular layer with elevated composition of protein containing halogenated amino acids and cross-linked via dityrosine linkages. The fibrous layer makes up the bulk of the tunic and is comprised primarily of helicoidally-ordered crystalline cellulose fibres with a lower protein content. The subcuticular zone directly beneath the surface contains much less organized cellulose fibres. Given current efforts to utilize biorenewable cellulose sources for the sustainable production of bio-inspired composites, these insights establish the tunic of H. roretzi as an exciting new archetype for extracting relevant design principles. STATEMENT OF SIGNIFICANCE: Tunicates are the only animals able to produce cellulose. They use this structural polysaccharide to build an exoskeleton called a tunic. Here, we investigate the composition and hierarchical structure of the tough tunic from the sea pineapple Halocynthia roretzi through a multiscale cross-disciplinary approach. The tunic of this species is a composite of cellulose and proteins with two distinct layers. At the surface is a thin sclerotized cuticular layer with a higher protein content containing halogenated amino acids and cross-linked via dityrosine linkages. The fibrous layer makes up the bulk of the tunic and is comprised of well-ordered cellulose fibres with a lower protein content. Given current efforts to utilize cellulose to produce advanced materials, the tunic of the sea pineapple provides a striking model for the design of bio-inspired cellulosic composites.

Shear-Induced ß-Crystallite Unfolding in Condensed Phase Nanodroplets Promotes Fiber Formation in a Biological Adhesive.

Natural materials provide an increasingly important role model for the development and processing of next-generation polymers. The velvet worm Euperipatoides rowelli hunts using a projectile, mechanoresponsive adhesive slime that rapidly and reversibly transitions into stiff glassy polymer fibers following shearing and drying. However, the molecular mechanism underlying this mechanoresponsive behavior is still unclear. Previous work showed the slime to be an emulsion of nanoscale charge-stabilized condensed droplets comprised primarily of large phosphorylated proteins, which under mechanical shear coalesce and self-organize into nano- and microfibrils that can be drawn into macroscopic fibers. Here, we utilize wide-angle X-ray diffraction and vibrational spectroscopy coupled with in situ shear deformation to explore the contribution of protein conformation and mechanical forces to the fiber formation process. Although previously believed to be unstructured, our findings indicate that the main phosphorylated protein component possesses a significant ß-crystalline structure in the storage phase and that shear-induced partial unfolding of the protein is a key first step in the rapid self-organization of nanodroplets into fibers. The insights gained here have relevance for sustainable production of advanced polymeric materials.

The Puzzle of the Walnut Shell: A Novel Cell Type with Interlocked Packing.

The outer protective shells of nuts can have remarkable toughness and strength, which are typically achieved by a layered arrangement of sclerenchyma cells and fibers with a polygonal form. Here, the tissue structure of walnut shells is analyzed in depth, revealing that the shells consist of a single, never reported cell type: the polylobate sclereid cells. These irregularly lobed cells with concave and convex parts are on average interlocked with 14 neighboring cells. The result is an intricate arrangement that cannot be disassembled when conceived as a 3D puzzle. Mechanical testing reveals a significantly higher ultimate tensile strength of the interlocked walnut cell tissue compared to the sclerenchyma tissue of a pine seed coat lacking the lobed cell structure. The higher strength value of the walnut shell is explained by the observation that the crack cannot simply detach intact cells but has to cut through the lobes due to the interlocking. Understanding the identified nutshell structure and its development will inspire biomimetic material design and packaging concepts. Furthermore, these unique unit cells might be of special interest for utilizing nutshells in terms of food waste valorization, considering that walnuts are the most widespread tree nuts in the world.

Artificial hagfish protein fibers with ultra-high and tunable stiffness.

Stiff fibers are used as reinforcing phases in a wide range of high-performance composite materials. Silk is one of the most widely studied bio-fibers, but alternative materials with specific advantages are also being explored. Among these, native hagfish (Eptatretus stoutii) slime thread is an attractive protein-based polymer. These threads consist of coiled-coil intermediate filaments (IFs) as nano-scale building blocks, which can be transformed into extended ß-sheet-containing chains upon draw-processing, resulting in fibers with impressive mechanical performance. Here, we report artificial hagfish threads produced by recombinant protein expression, which were subsequently self-assembled into coiled-coil nanofilaments, concentrated, and processed into ß-sheet-rich fibers by a "picking-up" method. These artificial fibers experienced mechanical performance enhancement during draw-processing. We exploited the lysine content to covalently cross-link the draw-processed fibers and obtained moduli values (E) in tension as high as ∼20 GPa, which is stiffer than most reported artificial proteinaceous materials.