Mesoscale Confinement in Bagworm Silk: A Hidden Structural Organization.

While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline ß-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider's dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Effects of RGD-fused silk fibroin in a solution format on fibroblast proliferation and collagen production.

BACKGROUND: Collagen production in fibroblasts is important for skin tissue repair. Cell-adhesive Arg-Gly-Asp (RGD) peptides immobilized on scaffolds stimulate fibroblast collagen production, but RGD peptides in solution exhibit opposite effects. Transgenic silkworm technology enables the design of fusion positions for RGD peptides in silk fibroin molecules. The effect of RGD-fused silk fibroin in solution on fibroblast cell activity remains unclear. OBJECTIVE: To clarify the effects of RGD peptides fused to silk fibroin heavy (H)-chain or light (L)-chain on fibroblast proliferation and collagen production when RGD-fused silk fibroin proteins were added to the culture medium. METHODS: Silk fibers with RGD-fused H-chains (H-RGD) or L-chains (L-RGD) were degummed, dissolved, and dialyzed to prepare H-RGD or L-RGD aqueous solutions, respectively. These solutions were added to the fibroblast medium, and their proliferation and collagen production were quantified. RESULTS: Both L- and H-RGD stimulated fibroblast proliferation at a similar level, even in a solution format, but L-RGD promoted fibroblast collagen production significantly, indicating the synergistic effect of the native H-chain and RGD-fused L-chain. CONCLUSION: RGD-fused silk fibroin in solution stimulated fibroblast proliferation and collagen production, depending on the fusion position of the peptides.

Immobilization of Arg-Gly-Asp peptides on silk fibroin via Gly-Ala-Gly-Ala-Gly-Ser sequences.

A simple method by which the functional peptide of Gly-Arg-Gly-Asp-Ser (GRGDS) is immobilized on the surface of silk fibroin (SF) films via Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS) sequences is proposed. GAGAGS, a repeating amino acid sequence in the crystal region of Bombyx mori SF, performs a key role in interacting with and immobilizing SF molecules. Immobilization by this proposed method involves no chemical reaction, thereby preserving the original properties of the SF molecule. The density of GRGDS peptides existing on SF film was found to be higher in the GAGAGS-bound type than in the non-GAGAGS-bound type. Furthermore, results showed that the amount of immobilized (GAGAGS)GRGDS peptide increased as the ß-sheet crystallization was promoted in the SF film. Fibroblasts, which adhered to the surface of the SF film, showed more extensibility because of the (GAGAGS)GRGDS immobilization, which suggests that the cell adhesion activity of RGD is functioning effectively.

Silk Fibroin Conjugated with Heparin Promotes Epithelialization and Wound Healing.

Silk fibroin (SF) has attracted attention as a base biomaterial that could be suitable in many applications because of its shape and structure. Highly functional SF has been developed to promote tissue regeneration with heparin conjugation. However, the hydrophobic three-dimensional structure of SF makes it difficult to bind to high-molecular-weight and hydrophilic compounds such as heparin. In this study, sufficient heparin modification was achieved using tyrosine residues as reaction points to improve cellular response. As it was considered that there was a trade-off between the improvement of water wettability and cell responsiveness induced by heparin modification, influences on the structure, and mechanical properties, the structure and physical properties of the SF conjugated with heparin were extensively evaluated. Results showed that increased amounts of heparin modification raised heparin content and water wettability on film surfaces even though SF formation was not inhibited. In addition, the proliferation of endothelial cells and fibroblasts were enhanced when a surface with sufficient heparin assumed its potential in assisting wound healing. This research emphasizes the importance of material design focusing on the crystal structure inherent in SF in the development of functionalized SF materials.

A study of ladder-like silk foothold for the locomotion of bagworms.

While walking on horizontal substrates, caterpillars skilfully engage all their legs, including three pairs of thoracic legs and a maximum of five pairs of prolegs, to move in a flexible wave-like motion. Such locomotory behaviours, represented by 'crawling' and 'inching' motions, have widely inspired the development of locomotion systems in soft robotics. However, bagworms are unable to use their prolegs for walking because these are always accommodated in a portable bag; thus, they are unable to walk using such general locomotory behaviours. Indeed, how they walk with only three pairs of thoracic legs is unknown at present. In this study, we show that bagworms construct a ladder-like foothold using their silk to walk without using prolegs. This enables them to walk not only on horizontal floor surfaces but also on wall and ceiling surfaces, even those with slippery or smooth surfaces. They construct the foothold by spinning a continuous silk thread in a zigzag manner and controlling the discharge of adhesive to attach the folded parts of the silk to a substrate. Discovery of this elaborate silk utilisation technique offers fresh insights into the diversity of silk use in lepidopteran larvae and provides potential designs for robot locomotion systems.

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

Genome-wide SNP marker discovery and phylogenetic analysis of mulberry varieties using double-digest restriction site-associated DNA sequencing.

There has been long taxonomic debate on mulberry species (genus Morus) because the classification of mulberry species has relied on morphological characteristics. Although attempts for classifying mulberry species using molecular markers have been performed, phylogenetic relationships among diploid mulberry species remain unclear. In this study, we aim to investigate the genetic relationship between 54 diploid mulberry varieties belonging to seven different Morus species (M. alba, M. indica, M. bombycis, M. acidosa, M. latifolia, M. kagayamae, and M. rotundiloba) and one unspecified Morus species ('Enbu') using genome-wide SNP discovery and phylogenetic analysis via double-digest restriction site-associated DNA sequencing (ddRAD-seq). Genome-wide 2229 homozygous SNPs of 54 mulberry varieties in the eight species were identified by ddRAD-seq. Results of the phylogenetic analysis identified only three clear monophyletic clades in two Japanese native species, M. acidosa and M. kagayamae, which are found on different geographically isolated islands and a Thai species, M. rotundiloba, whereas the other species were non-monophyletic. Varieties of M. bombycis, another Japanese native species, were roughly classified into three groups. Of these, two M. bombycis groups were monophyletic with M. acidosa and M. kagayamae, respectively, while another M. bombycis group was not monophyletic. Varieties of M. indica, an Indian native species, were classified into two different monophyletic clades. Of these, one clade was clearly monophyletic with an indigenous variety in Kenya, 'Enbu', while another clade was monophyletic with M. rotundiloba and one M. latifolia variety. There were no clear monophyletic clades within M. alba and M. latifolia varieties, which could be a result of several hybridization events after their introductions from China to Japan. Our results suggested that it was difficult to clearly classify the hybridized mulberry varieties even with genome-wide DNA markers. In addition to phylogenetic analysis, we also evaluated morphological characteristics of mulberry leaves for each variety. The results of morphological evaluation indicated that leaf tip ratio may correlate to genetic difference among the two M. bombycis groups in monophyletic clades and another M. bombycis group in non-monophyletic clades. These results suggested that leaf tip ratio might be used for evaluating hybridization of M. bombycis varieties. Over all, our results may provide new insights into taxonomic debate of mulberry species.

Structure Water-Solubility Relationship in α-Helix-Rich Films Cast from Aqueous and 1,1,1,3,3,3-Hexafluoro-2-Propanol Solutions of S. c. ricini Silk Fibroin.

Silk fibroin (SF) produced by the domesticated wild silkworm, Samia cynthia ricini (S. c. ricini) is attracting increasing interest owing to its unique mechanical properties, biocompatibility, and abundance in nature. However, its utilization is limited, largely due to lack of appropriate processing strategies. Various strategies have been assessed to regenerate cocoon SF, as well as the use of aqueous liquid fibroin (LFaq) prepared by dissolution of silk dope obtained from the silk glands of mature silkworm larvae in water. However, films cast from these fibroin solutions in water or organic solvents are often water-soluble and require post-treatment to render them water-stable. Here, we present a strategy for fabrication of water-stable films from S. c. ricini silk gland fibroin (SGF) without post-treatment. Aqueous ethanol induced gelation of fibroin in the posterior silk glands (PSG), enabling its separation from the rest of the silk gland. When dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), the SGF-gel gave a solution from which a transparent, flexible, and water-insoluble film (SGFHFIP) was cast. Detailed structural characterization of the SGFHFIP as-cast film was carried out and compared to a conventional, water-soluble film cast from LFaq. FTIR and 13C solid-state NMR analyses revealed both cast films to be α-helix-rich. However, gelation of SGF induced by the 40%-EtOH-treatment resulted in an imperfect ß-sheet structure. As a result, the SGF-gel was soluble in HFIP, but some ß-sheet structural memory remains, and the SGFHFIP as-cast film obtained has some ß-sheet content which renders it water-resistant. These results reveal a structure water-solubility relationship in S. c. ricini SF films that may offer useful insights towards tunable fabrication of novel biomaterials. A plausible model of the mechanism that leads to the difference in water resistance of the two kinds of α-helix-rich films is proposed.

Aggregation State of Residual α-Helices and Their Influence on Physical Properties of S. c. ricini Native Fiber.

Formation of the α-helical conformation in the poly-l-alanine (PA) sequence regions, subsequent structural transition to ß-sheet during natural spinning, and presence of residual α-helices in Samia cynthia ricini (S. c. ricini) native silk fiber have been experimentally proven. However, the aggregation state of the residual α-helices, and their influence on the mechanical deformation behavior in native fiber remain unclear. Here we show that the α-helices form an ordered aggregation state with a hexagonal packing in the aqueous solution, some of which remain during natural spinning. X-ray scattering and differential scanning calorimetry (DSC) analyses revealed occurrence of a structural transition of the residual α-helices to the ß-sheet structure, accompanied by disappearance of the plateau region in the force-strain curve, due to heat-treatment at ~220 °C. On the basis of X-ray scattering before and after tensile stretching of S. c. ricini native silk, a direct connection between the plateau region and the α-helix to ß-sheet structural transition was confirmed. Our findings demonstrate the importance of the PA sequence regions in fiber structure formation and their influence on the tensile deformation behavior of S. c. ricini silk, features believed to be essentially similar in other saturniid silks. We strongly believe the residual ordered α-helices to be strategically and systematically designed by S. c. ricini silkworms to impart flexibility in native silk fiber. We anticipate that these knowledge forms a basis for fruitful strategies in the design and development of amino acid sequences for artificial silks with desired mechanical properties.

A study of the extraordinarily strong and tough silk produced by bagworms.

Global ecological damage has heightened the demand for silk as 'a structural material made from sustainable resources'. Scientists have earnestly searched for stronger and tougher silks. Bagworm silk might be a promising candidate considering its superior capacity to dangle a heavy weight, summed up by the weights of the larva and its house. However, detailed mechanical and structural studies on bagworm silks have been lacking. Herein, we show the superior potential of the silk produced by Japan's largest bagworm, Eumeta variegata. This bagworm silk is extraordinarily strong and tough, and its tensile deformation behaviour is quite elastic. The outstanding mechanical property is the result of a highly ordered hierarchical structure, which remains unchanged until fracture. Our findings demonstrate how the hierarchical structure of silk proteins plays an important role in the mechanical property of silk fibres.

Transdermal delivery of 40-nm silk fibroin nanoparticles.

Transdermal administration of drugs improves their bioavailability and is capable of systemic and local treatment. To improve the skin permeability of drugs, nano-sized systems have attracted attention as drug carriers for transdermal drug delivery system. We considered that silk fibroin composed of a crystalline region with many hydrophobic amino acids and an amorphous region with many hydrophilic amino acids was useful as a carrier for transdermal administration of a drug because of the balance between hydrophilicity and hydrophobicity. In this study, silk fibroin nanoparticles with mean volume diameters of 42.3 nm were successfully prepared, and storage stability was confirmed by storing the nanoparticle suspension at 4, 32, and 37 °C for a week. At any storage temperature, the mean volume diameter and standard deviation were stable. The polydispersity indexes were 0.19-0.23, and no specific trends were observed. Then, to investigate the transdermal delivery route of the silk fibroin nanoparticles, skin permeability in vivo was evaluated using mice. Six hours after administration, fluorescent substances were observed in the dermis in addition to the stratum corneum, hair follicles and the epidermis around them. This result indicated that fibroin nanoparticles with the mean volume diameter of 40-nm penetrated the stratum corneum and was delivered deep into the skin. Therefore, it was suggested that small nanoparticles prepared using silk fibroin are useful for drug delivery to the dermis.

Conformation change of hornet silk proteins in the solid phase in response to external stimulation.

Hornet silks adopt a variety of morphology such as fibers, sponge, films, and gels depending on the preparation methods. We have studied the conformation change of hornet silk proteins (Vespa mandarina) as regenerated films, using chiroptical spectrophotometer universal chiroptical spectrophotometer 1, which can measure true circular dichroism spectra without artifact signals that are intrinsic to solid-state samples. The spectra showed that the proteins in films alter the conformation rapidly from the α-helix to the coiled coil and then to a ß-sheet structure in response to heat/moisture treatment, but the transformation stopped at the coiled coil state when the sample was soaked in EtOH/water solution. Water is required for the α-helix to the coiled coil transition, and extra energy is required for the further transition to a ß-sheet structure. This is the first successful circular dichroism study of fibril silk proteins to follow the conformation changes in the solid state. This work shows that proteins can undergo conformational changes easily even in the solid phase in response to external stimuli, and this can be traced by solid-phase-feasible chiroptical spectrophotometers. Application of unnatural stress to proteins gives valuable insights into their structure and characteristics.

Genome Editing Advances the Structural Study of Silk.

We first applied the genome edited silkworm silk (GE-silk) to interpret X-ray fiber diagram, and implied a great potential for the application of genome editing technology to the structural study of silk. The origin of a weak meridional layer-line streak with a spacing of ∼21 Å, observed in the X-ray fiber diagram of Bombyx mori silkworm silk, has been widely believed but not experimentally proven to be a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in a hexapeptide repeating unit -G-A-G-A-G-S-. The above hypothesis was experimentally demonstrated from X-ray measurements of GE-silk.

Compatibility Evaluation of Non-Woven Sheet Composite of Silk Fibroin and Polyurethane in the Wet State.

SF/polyurethane composite non-woven sheet was fabricated to evaluate the cardiovascular tissue engineering materials in the wet state. The compatibility and microstructure analyses were carried out on the fabricated SF/polyurethane composite non-woven sheet by thermal analysis and solid-state NMR analysis in the wet state. To evaluate the modulus of elasticity, a tensile test was performed and supported with dynamic viscoelasticity and mechanical analysis. Results showed that SF/polyurethane composites form domains within the non-woven sheet and are in a finely dispersed state while maintaining their structures at a scale of several tens of nm. Moreover, an increase of the loss tangent with low elastic modulus proved that a micromolecular interaction occurs between silk fibroin (SF) and polyurethane molecules.

Transformation of Coiled α-Helices into Cross-ß-Sheets Superstructure.

The fibrous silk produced by bees, wasps, ants, or hornets is known to form a four-strand α-helical coiled coil superstructure. We have succeeded in showing the formation of this coiled coil structure not only in natural fibers, but also in artificial films made of regenerated silk of the hornet Vespa simillima xanthoptera using wide- and small-angle X-ray scatterings and polarized Fourier transform infrared spectroscopy. On the basis of time-resolved simultaneous synchrotron X-ray scattering observations for in situ monitoring of the structural changes in regenerated silk material during tensile deformation, we have shown that the application of tensile force under appropriate conditions induces a transition from the coiled α-helices to a cross-ß-sheet superstructure. The four-stranded tertiary superstructure remains unchanged during this process. It has also been shown that the amorphous protein chains in the regenerated silk material are transformed into conventional ß-sheet arrangements with varying orientation.

Fabrication Scheme for Obtaining Transparent, Flexible, and Water-Insoluble Silk Films from Apparently Dissolved Silk-Gland Fibroin of Bombyx mori Silkworm.

Films from silk fibroin protein are one of the most promising biomaterials because of their exquisite balance between mechanical properties and biocompatibility. Numerous schemes have been proposed for processing fibroin film, utilizing liquid silk fibroin (LSF) or regenerated silk fibroin (RSF). The films cast from LSF or RSF in the solution state are water-soluble, and therefore require postproduction treatment inducing ß-sheet formation, to render them insoluble in water. Many kinds of postproduction treatments, using alcohol-water solution, water vapor, or controlled temperature, have been developed. However, the tuning and reproducibility of such treatments are quite sensitive and frequently render the fibroin films less flexible or even brittle because of the formation of an over content of ß-sheet. To overcome this, we developed a novel scheme for fibroin processing using silk-gland fibroin (SGF). The essence of this scheme is to create a softly solidified fibroin-gel state of the silk glands with an imperfect ß-sheet structure, by treating them with an ethanol/water mixture. Such a fibroin gel was found to dissolve in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The SGF film cast from the HFIP solution shows a flexible and water-insoluble nature with high reproducibility. In addition to this improvement, the SGF film produced by this method contains a significantly low level of residual HFIP molecules compared to the traditional RSF films prepared from an HFIP solution. The mechanism underlying these advantageous characteristics was investigated from the structural viewpoint, by using techniques such as 13C solid-state NMR, differential scanning calorimetry, and wide-angle X-ray diffraction.

Silk fibroin sponges with cell growth-promoting activity induced by genetically fused basic fibroblast growth factor.

Transgenic silkworm technology has enabled the biological properties of silk fibroin protein to be altered by fusion to recombinant bioactive proteins. However, few studies have reported the fabrication of genetically modified fibroin proteins into three-dimensional spongy structures to serve as scaffolds for tissue engineering. We generated a transgenic silkworm strain that produces fibroin fused to basic fibroblast growth factor (bFGF) and processed the fibroin into a spongy structure using a simple freeze/thaw method. NIH3T3 mouse embryonic fibroblasts grown on bFGF-fused fibroin sponges proliferated and spread out well, showing half the population doubling time of cells cultured on wild-type fibroin sponges. Furthermore, the number of primary rabbit articular chondrocytes growing on bFGF-fused fibroin sponges was around five-times higher than that of the wild-type control at 3-days post cell-seeding. As the physical properties of wild-type and bFGF-fused fibroin sponges were almost identical, it is suggested that bFGF fused to fibroin retained its biological activity, even after the bFGF-fused fibroin was fabricated into the spongy structure. The bFGF-fused fibroin sponge has the potential for widespread application in the field of tissue engineering, and the method of fabricating this structure could be applicable to other recombinant bioactive fibroin proteins.

Influence of pH, temperature, and concentration on stabilization of aqueous hornet silk solution and fabrication of salt-free materials.

We found that an aqueous solution of silk from cocoons produced by hornet larvae (hornet silk) can be obtained when the solution is adjusted to basic conditions of pH > 9.2. It is known that native hornet cocoons can be dissolved in concentrated aqueous solution of salts, such as lithium bromide (LiBr) and calcium chloride (CaCl2). Upon the removal of these salts from solution by dialysis, solidification, gelation, or sedimentation of hornet silk is known to occur. In the present study, under basic conditions, however, no such solidification occurred, even after salt removal. In this study, ammonia was used for alkalization of solution because it is volatilized during the casting process and pure hornet silk materials can be obtained after drying. The effects of the concentrations of hornet silk and ammonia, as well as dialysis temperature, on preventing gelation during dialysis were investigated. Dialysis conditions that limit the degradation of hornet silk by hydrolysis in alkali solution were identified. Moreover, casting conditions to prepare flexible and transparent hornet silk film from aqueous ammonia solution were optimized. Molecular structural analysis of hornet silk in aqueous ammonia solution and cast film indicated the formation of α-helix conformations.

Stabilization of viruses by encapsulation in silk proteins.

Viruses are important for a range of modern day applications. However, their utility is limited by their susceptibility to temperature degradation. In this study, we report a simple system to compare the ability of different dried protein films to stabilize viruses against exposure to elevated temperatures. Films from each of three different silks, silkworm, honeybee silk and hornet silk, stabilized entrapped viruses at 37 °C better than films of albumin from bovine serum (BSA) and all four proteins provided substantially more stabilization than no protein controls. A comparison of the molecular structure of the silks and BSA films showed no correlation between the ability of the proteins to stabilize the virus and the secondary structure of the protein in the films. The mechanism of stabilization is discussed and a hypothesis is suggested to explain the superior performance of the silk proteins.