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Articular cartilage degradation due to injury, disease and aging is a common clinical issue as current regenerative therapies are unable to fully replicate the complex microenvironment of the native tissue which, being avascular, is featured by very low ability to self-regenerate. The extracellular matrix (ECM), constituting almost 90% of the entire tissue, plays a critical role in its function and resistance to compressive forces. In this context, the current tissue engineering strategies are only partially effective in restoring the biology and function of the native tissue. A main issue in tissue regeneration is treatment failure due to scarce integration of the engineered construct, often following a gradual detachment of the graft. In this scenario, we aimed to create an adhesive patch able to adequately support cartilage regeneration as a promising tool for the treatment of cartilage injuries and diseases. For this, we produced an engineered construct composed of decellularized ECM (dECM) obtained from horse joint cartilage, to support tissue regeneration, coupled with a Silk-Elastin-Like Proteins (SELP) hydrogel, which acts as a biological glue, to guarantee an adequate adherence to the host tissue. Following the production of the two biomaterials we characterized them by assessing: 1) dECM morphological, chemical, and ultrastructural features along with its capability to support chondrocyte proliferation, specific marker expression and ECM synthesis; 2) SELP microarchitecture, cytocompatibility and mechanical properties. Our results demonstrated that both materials hold unique properties suitable to be exploited to produce a tailored microenvironment to support cell growth and differentiation providing a proof of concept concerning the in vitro biological and mechanical efficacy of the construct. The SELP hydrogel displayed a very interesting physical behavior due to its high degree of resistance to mechanical stress, which is generally associated with physiological mechanical load during locomotion. Intriguingly, the shear-thinning behavior of the hydrogel may also make it suitable to be applied and spread over non-homogeneous surfaces, therefore, we hypothesize that the hybrid biomaterial proposed may be a real asset in the treatment of cartilage defects and injuries., Competing Interests: Declaration of Competing Interest The authors of the manuscript “Biomimetic approach for an articular cartilage patch: combination of decellularized cartilage matrix and silk-elastin-like-protein (SELP) hydrogel” declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Wearable sweat electrochemical sensors have attracted wide attention due to their advantages of non-invasive, portable, real-time monitoring, etc. However, existing sensors still have some problems with efficient sweat collection. Microfluidic channel technology and electrospinning technology are commonly used to collect sweat efficiently, but there are some limitations such as complex channel design and multiple spinning parameters. Besides, existing sensors are mostly based on flexible polymers, such as, PET, PDMS, PI and PI, which have limited wearability and permeability. Based on the above, all fabric and dual-function flexible wearable sweat electrochemical sensor is proposed in this paper. This sensor uses fabric as the raw material to implement the directional transport of sweat and the multi-component integrated detection dual functions. Meanwhile, the high-efficiency collection of sweat is obtained by a Janus fabric, wherein one side of the selected silk is superhydrophobic graft treated and the other side is hydrophilic plasma treated. Therefore, the resulting Janus fabric can effectively transfer sweat from the skin side to the electrode, and the minimum sweat droplet can reach 0.2 μL to achieve micro-volume collection. Besides, the patterned sensor, made of silk-based carbon cloth, is fabricated using a simple laser engraving, which could detect Na + , pH, and glucose instantaneously. As a result, these proposed sensors can achieve good sensing performance and high-efficiency sweat collection dual functionality; moreover, it has good flexibility and comfortable wearability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The late 5th instar caterpillar of the cecropia silk moth (Hyalophora cecropia) spins a silken cocoon with a distinct, multilayered architecture. The cocoon construction program, first described by the seminal work of Van der Kloot and Williams, consists of a highly ordered sequence of events. We perform behavioral experiments to re-evaluate the original cecropia work, which hypothesized that the length of silk that passes through the spinneret controls the orderly execution of each of the discrete events of cocoon spinning. We confirm and extend by three-dimensional scanning and quantitative measurements of silk weights that if cocoon construction is interrupted, upon re-spinning, the caterpillar continues the cocoon program from where it left off. We also confirm and extend by quantitative measurements of silk weights that cecropia caterpillars will not bypass any of the sections of the cocoon during the construction process, even if presented with a pre-spun section of a cocoon spun by another caterpillar. Blocking silk output inhibits caterpillars from performing normal spinning behaviors used for cocoon construction. Surprisingly, unblocking silk output 24-hr later did not restart the cocoon construction program, suggesting the involvement of a temporally-defined interval timer. We confirm with surgical reductions of the silk glands that it is the length of silk itself that matters, rather than the total amount of silk extracted by individuals. We used scanning electron microscopy to directly show that either mono- or dual-filament silk (i.e., equal silk lengths but which vary in their total amount of silk extracted) can be used to construct equivalent cocoons of normal size and that contain the relevant layers. We propose that our findings, taken together with the results of prior studies, strongly support the hypothesis that the caterpillar uses a silk "odometer" to measure the length of silk extracted during cocoon construction but does so in a temporally regulated manner. We further postulate that our examination of the anatomy of the silk spinning apparatus and ablating spinneret sensory output provides evidence that silk length measurement occurs upstream of output from the spinneret., Competing Interests: The authors have declared that no competing interests exist.

In recent years, increasing attention has been paid to the origin, transmission and communication of silk. However, this is still an unsolved mystery in archaeology. The identification of silk-producing species, especially silk produced by Bombyx mori (B. mori) and Antheraea pernyi (A. pernyi), is of key significance to address this challenge. In this study, two innovative methods, i.e. immunology and proteomics, were proposed and successfully established for the species identification of silks. ELISAs result demonstrated that the two prepared antibodies exhibited high sensitivity and specificity in distinguishing B. mori and A. pernyi silk. No cross-reactivity with each other was observed. Moreover, biomarkers were obtained for Bombyx and Antheraea through proteomic analysis. It was also confirmed that the biomarkers were suitable for identifying the species that produced a given silk sample. Compared with conventional methods for distinguishing silk species, immunological and proteomics techniques used in tandem can provide intact information and have the potential to provide accurate and reliable information for species identification.

The silkworm cocoon colour has attracted researchers involved in genetics, physiology and ecology for a long time. 'Ryokuken' cocoons are yellowish green in colour due to unusual flavonoids, prolinylflavonols, while 'Sasamayu' cocoons are light green and contain only simple flavonol glucosides. We found a novel gene associated with the cocoon colour change resulting from a change in flavonoid composition and named it Lg (light green cocoon). In the middle silk glands of the + Lg /+ Lg larvae, 1-pyrroline-5-carboxylic acid (P5C) was found to accumulate due to a decrease in the activity of pyrroline-5-carboxylate reductase (P5CR), an enzyme reducing P5C to proline. Sequence analysis of BmP5CR1, the candidate gene for Lg, revealed a 1.9 kb insertion and a 4 bp deletion within the 1st intron, a 97 bp deletion within the 4th intron, and a > 300 bp insertion within the 3'-UTR, in addition to two amino acid changes on exons 3 and 4 in + Lg /+ Lg compared to Lg/Lg. Decreased expression of BmP5CR1 was observed in all of the investigated tissues, including the middle silk glands in + Lg /+ Lg , which was probably caused by structural changes in the intronic regions of BmP5CR1. Furthermore, a BmP5CR1 knockout strain exhibited a yellowish green cocoon with the formation of prolinylflavonols. These results indicate that the yellowish green cocoon is produced by a BmP5CR1 deficiency. To our knowledge, this is the first report showing that the defect of an enzyme associated with intermediate metabolism promotes the conjugation of phytochemicals derived from foods with endogenously accumulating metabolites in animal tissues.

Recently, much interest has been paid to the separation of silk produced by Bombyx mori from silk produced by other species and tracing the beginnings of silk cultivation from wild silk exploitation. In this paper, significant differences between silks from Bombyx mori and other species were found by microscopy and spectroscopy, such as morphology, secondary structure, and amino acid composition. For further accurate identification, a diagnostic antibody was designed by comparing the peptide sequences of silks produced by Bombyx mori and other species. The results of the noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) indicated that the antibody that showed good sensitivity and high specificity can definitely discern silk produced by Bombyx mori from silk produced by wild species. Thus, the antibody-based immunoassay has the potential to be a powerful tool for tracing the beginnings of silk cultivation. In addition, combining the sensitive, specific, and convenient ELISA technology with other conventional methods can provide more in-depth and accurate information for species identification.

The classification and conservation of ancient artworks (belonging to collections) is of important cultural, historical, and economic concern. However, ancient textiles often display structural damage that renders them fragile and unsuitable for exhibition. One of the most common types of damage is linked to erroneous restoration treatments, among which the application of glues to consolidate cuts. Harsh strategies, such as mechanical or chemical treatments, are not suitable since they can cause further impairment of the fabric, whereas mild approaches, like wet cleaning, are often ineffective, as also demonstrated by the present study. Here, we have explored the possibility of using gellan-immobilized enzymes of bacterial origin (Bacillus alpha-amylase) to obtain a satisfactory starch removal from a damaged archaeological tunic-shroud from the Turin Egyptian Museum (Italy), without altering the original yarns or textile fibers. This method, already applied to clean casein-damaged wall paintings, as well as cotton, silk, and linen fabrics, has proved to be optimal for the treatment of a wool burial shroud and to be able to definitively solve fragile textile restoration problems. Moreover, efforts have been made to obtain insights into the artwork: a multidisciplinary approach has allowed to obtain a correct chronological attribution (radiocarbon dating) and fabric fiber characterization (SEM-EDX) as well as shed light on the colored parts and dark stains (FORS+IRFC and XRF). Finally, the evaluation of the type of glue, by Fourier transform infrared spectroscopy, has suggested the best enzyme for glue removal. These results have demonstrated that a mild bio-based approach is a successful tool for the treatment of archaeological textiles in critical conditions.

Building behaviour in animals extends biological functions beyond bodies. Many studies have emphasized the role of behavioural programmes, physiology and extrinsic factors for the structure and function of buildings. Structure attachments associated with animal constructions offer yet unrealized research opportunities. Spiders build a variety of one- to three-dimensional structures from silk fibres. The evolution of economic web shapes as a key for ecological success in spiders has been related to the emergence of high performance silks and thread coating glues. However, the role of thread anchorages has been widely neglected in those models. Here, we show that orb-web (Araneidae) and hunting spiders (Sparassidae) use different silk application patterns that determine the structure and robustness of the joint in silk thread anchorages. Silk anchorages of orb-web spiders show a greater robustness against different loading situations, whereas the silk anchorages of hunting spiders have their highest pull-off resistance when loaded parallel to the substrate along the direction of dragline spinning. This suggests that the behavioural 'printing' of silk into attachment discs along with spinneret morphology was a prerequisite for the evolution of extended silk use in a three-dimensional space. This highlights the ecological role of attachments in the evolution of animal architectures., (© 2017 The Author(s).)

One of the key problems hindering skin repair is the deficiency of dermal vascularization and difficulty of epidermis regeneration, which makes it challenging to fabricate scaffolds that can biologically fulfill the requirements for skin regeneration. To overcome this problem, three-dimensional printing was used to fabricate a gelatin-sulfonated silk composite scaffold that was incorporated with basic fibroblast growth factor 2 (FGF-2) through binding with a sulfonic acid group (SO 3 ) (3DG-SF-SO 3 -FGF). The efficacy and mechanism by which the 3DG-SF-SO 3 -FGF scaffolds promote skin regeneration were investigated both within in vitro cell culture and in vivo with a full-thickness skin defect model. The histological results showed that the gelatin-sulfonated silk composite scaffolds promoted granulation, and that incorporation of FGF-2 significantly enhanced the regeneration of skin-like tissues after implantation in rat skin defects for 14 and 28 days. Further investigations demonstrated that 3DG-SF-SO 3 -FGF scaffolds might stimulate dermal vascularization. These findings thus suggest that incorporation of FGF-2 into the 3D printed scaffolds is a viable strategy for enhancing skin regeneration.

The silkworm spinneret is an important tissue for silk fibrillogenesis and spinning. All biochemical processes during silk fibrillogenesis are correlated with silk properties. Understanding the role of spinneret in silk fibrillogenesis may help to reveal the mechanism of silk fibrillogenesis as well as improve silk quality for commercial purposes. Thus, we profiled the proteome of silkworm spinneret. A total of 1572 proteins and 232 differential abundance proteins were identified. Silk fibrillogenesis-related proteins, such as cuticle proteins, ion-transporting proteins, muscular proteins, and energy metabolic proteins, were abundant in spinneret. Metabolic pathway and GO enrichment analyses revealed that the identified proteins were involved in energy metabolism, chitin binding, and cuticle construction. Active energy metabolism may provide abundant energy for the muscle contraction as well as ion and water exchange. The chitin binding and cuticle construction process may provide sufficient shear forces for silk formation. Our data suggest that silkworm spinneret provides a suitable physiological and biochemical environment for silk fibrillogenesis. These proteins are potential targets for improving silk quality in the silk industry. Data are available via ProteomeXchange with identifier PXD004455., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In light of recent focus on the behaviour of the natural structures for revolutionary technological growth, spider web seems to have seized considerable attention of product designer due to its amazing behaviour. In present work, mechanism behind the structural integrity of the spider web along with the materialistic analysis of its constituent silk threads has been extensively investigated. The nanoindentation tool both in static and dynamic mode has been utilized for complete analysis of the mechanical behaviour of the spiral and radial threads separately. Both the average elastic modulus and hardness of the radial silk thread is higher than the spiral silk thread which reveals the radial silk thread is the major structural component of the web. The sustainability of spider webs under storm, windy conditions and during the impact of pray has been investigated under dynamic conditions. The radial silk thread exhibits elastic like response and the spiral silk thread exhibits viscous like response in a wide frequency range (1-200Hz). The damping characteristic of the radial and spiral silk threads, an important parameter to investigate the energy dissipation properties of the materials has also been investigated in windy conditions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The unique combination of great stiffness, strength, and extensibility makes spider major ampullate (MA) silk desirable for various biomimetic and synthetic applications. Intensive research on the genetics, biochemistry, and biomechanics of this material has facilitated a thorough understanding of its properties at various levels. Nevertheless, methods such as cloning, recombination, and electrospinning have not successfully produced materials with properties as impressive as those of spider silk. It is nevertheless becoming clear that silk properties are a consequence of whole-organism interactions with the environment in addition to genetic expression, gland biochemistry, and spinning processes. Here we assimilate the research done and assess the techniques used to determine distinct forms of spider silk chemical and physical property variability. We suggest that more research should focus on testing hypotheses that explain spider silk property variations in ecological and evolutionary contexts.

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like (TIL)-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens., (© 2015 Institute of Zoology, Chinese Academy of Sciences.)

Rationale: The δ(2) H values of some nitrogen-containing organic compounds measured by High-Temperature Conversion (HTC) with a glassy carbon reactor have been shown to be inaccurate. A probable explanation for these analytical inaccuracies is the formation of HCN, allowing some hydrogen atoms to escape isotope ratio measurement. We assess this isotopic effect in sample types commonly used for (paleo)ecological, environmental, archaeological, and forensic investigations., Methods: The δ(2) HVSMOW-SLAP values and mass fraction H using a factory-recommended glassy carbon HTC reactor packing were compared with those obtained from using two Cr-containing reactor packings for a variety of N-containing substances, including amino acids, collagen, hair, and silk., Results: δ(2) HVSMOW-SLAP values and mass fraction H differed by reactor packing for most, but not all, N-containing samples. The δ(2) HVSMOW-SLAP difference was 10-11 ‰ for modern collagen and 12-14 ‰ for hair, demonstrating that reactor configuration is important for these proteins, and that the use of a chromium-packed reactor may be desirable. In contrast, Bombyx mori cocoon (silk) δ(2) HVSMOW-SLAP values did not differ with reactor type. In general, δ(2) HVSMOW-SLAP and mass fraction H differences by reactor packing increased with mass fraction nitrogen in the sample. With the Cr-packed reactor hydrogen mass fractions were at theoretically expected values, while the glassy carbon reactor produced lower yields of hydrogen., Conclusions: The protein and amino acid δ(2) HVSMOW-SLAP values measured by factory-recommended online HTC methods differ from those from Cr-containing reactor packing. The magnitude of the differences is variable with sample type; the molecular structure and diagenetic history of each sample may be important. Careful attention to this effect is therefore recommended for the δ(2) H measurement for all nitrogen-containing analytes. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Predators have been shown to alter their foraging as a regulatory response to recent feeding history, but it remains unknown whether trap building predators modulate their traps similarly as a regulatory strategy. Here we fed the orb web spider Nephila pilipes either live crickets, dead crickets with webs stimulated by flies, or dead crickets without web stimulation, over 21 days to enforce spiders to differentially extract nutrients from a single prey source. In addition to the nutrients extracted we measured web architectures, silk tensile properties, silk amino acid compositions, and web tension after each feeding round. We then plotted web and silk "performance landscapes" across nutrient space. The landscapes had multiple peaks and troughs for each web and silk performance parameter. The findings suggest that N. pilipes plastically adjusts the chemical and physical properties of their web and silk in accordance with its nutritional history. Our study expands the application of the geometric framework foraging model to include a type of predatory trap. Whether it can be applied to other predatory traps requires further testing.

Arthropods face several key challenges in processing concentrated feedstocks of proteins (silk dope) into solid, semi-crystalline silk fibres. Strikingly, independently evolved lineages of silk-producing organisms have converged on the use of liquid crystal intermediates (mesophases) to reduce the viscosity of silk dope and assist the formation of supramolecular structure. However, the exact nature of the liquid-crystal-forming-units (mesogens) in silk dope, and the relationship between liquid crystallinity, protein structure and silk processing is yet to be fully elucidated. In this review, we focus on emerging differences in this area between the canonical silks containing extended-β-sheets made by silkworms and spiders, and 'non-canonical' silks made by other insect taxa in which the final crystallites are coiled-coils, collagen helices or cross-β-sheets. We compared the amino acid sequences and processing of natural, regenerated and recombinant silk proteins, finding that canonical and non-canonical silk proteins show marked differences in length, architecture, amino acid content and protein folding. Canonical silk proteins are long, flexible in solution and amphipathic; these features allow them both to form large, micelle-like mesogens in solution, and to transition to a crystallite-containing form due to mechanical deformation near the liquid-solid transition. By contrast, non-canonical silk proteins are short and have rod or lath-like structures that are well suited to act both as mesogens and as crystallites without a major intervening phase transition. Given many non-canonical silk proteins can be produced at high yield in E. coli, and that mesophase formation is a versatile way to direct numerous kinds of supramolecular structure, further elucidation of the natural processing of non-canonical silk proteins may to lead to new developments in the production of advanced protein materials., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

The demand for methods and technologies capable of rapid, inexpensive and continuous monitoring of health status or exposure to environmental pollutants persists. In this work, the development of novel surface-enhanced Raman spectroscopy (SERS) substrates from metal-coated silk fabric, known as zari, presents the potential for SERS substrates to be incorporated into clothing and other textiles for the routine monitoring of important analytes, such as disease biomarkers or environmental pollutants. Characterization of the zari fabric was completed using scanning electron microscopy, energy dispersive X-ray analysis and Raman spectroscopy. Silver nanoparticles (AgNPs) were prepared, characterized by transmission electron microscopy and UV-vis spectroscopy, and used to treat fabric samples by incubation, drop-coating and in situ synthesis. The quality of the treated fabric was evaluated by collecting the SERS signal of 4,4'-bipyridine on these substrates. When AgNPs were drop-coated on the fabric, sensitive and reproducible substrates were obtained. Adenine was selected as a second probe molecule, because it dominates the SERS signal of DNA, which is an important class of disease biomarker, particularly for pathogens such as Plasmodium spp. and Mycobacterium tuberculosis. Excellent signal enhancement could be achieved on these affordable substrates, suggesting that the developed fabric chips have the potential for expanding the use of SERS as a diagnostic and environmental monitoring tool for application in wearable sensor technologies.

Cultural fibrous material includes both important categories, i. e. textile and paper, consisting of precious cultural materials in museum, such as costume, painting, and manuscript. In recent years more and more connoisseur and conservator's concerns are, through nondestructive method, the authenticity and the ageing identification of these cultural relics especially made from fragile materials. In this research, we used attenuated total reflection infrared spectroscopy to identify five traditional textile fibers, alongside cotton, linen, wool, mulberry silk and tussah silk, and another five paper fibers alongside straw, wheat straw, long qisong, Chinese alpine rush and mulberry bar, which are commonly used for making Chinese traditional xuan paper. The research result showed that the animal fiber (wool, mulberry silk and tussah silk) and plant fiber (cotton and linen) were easier to be distinguished by comparing the peaks at 3 280 cm-1 belonging to NH stretching vibration and a serious peaks related to amide I to amide III. In the spectrum of wool, the peak at 1 076 cm-1 was assigned to the S-O stretching vibration absorption of cystine in wool structure and can be used to tell wool from silk. The spectrum of mulberry silk and tussah silk seems somewhat difficult to be identified, as well as the spectrum of cotton and linen. Five rural paper fibers all have obvious characteristic peaks at 3 330, 2 900 cm-1 which are related to OH and CH stretching vibration. In the fingerprint wavenumber range of 1 600 - 800 cm, the similar peaks also appeared at 1 370, 1 320 cm-1 and 1 162, 1 050 cm-1, both group peaks respectively are related to CH and CO vibration in the structure of cellulose and hemicellulose in paper fibers. Although there is more similarity of the infrared spectroscopy of these 5 paper fibers, some tiny difference in absorbance also can be found at 3 300 cm-1 and in the fingerprint range at 1 332, 1 203, and 1 050 cm-1 which are related to C-O-C vibration in cellulose. Moreover, in order to explore direct and simple method to identify different materials with similar spectrum,. the principal component analysis (PCA) was applied to separate cotton and linen, mulberry silk and tussah silk, as well as five paper fibers. To eliminate and reduce the spectral scattering caused by sample uneven surface roughness, the multiplicative scatter correction (MSC) has been applied based on total spectral data. The result showed that the score plot using the first two principal components can effectively categorize both group textiles of cotton and linen, as well as mulberry silk and tussah silk, and they have similar chemical structure. For five paper fibers, the PCA was applied in different spectral range (918-550, 1 280-918, 1 700-1 280 and 3 800-2 800 cm-1), and the best result appeared in the range from 3 800 to 2 800 cm-1, in which the five paper fibers can be well categorized. This research showed that infrared spectroscopy combined with principal component analysis has great potential advantage on identifying fibrous materials with similar structure.

N-hexane neuropathy is an occupational disease caused by exposure to n-hexane, which is used as a solvent in silk screen printing. Here, we describe a 35-year-old man, a silk screen printer by profession, who presented with dizziness, distal swelling of both lower limbs for 10 months and tingling and burning sensation in both feet for 9.5 months along with cold allodynia. The patient had normal results of a motor and sensory system examination, apart from an impaired temperature sense. Nerve conduction tests showed a conduction block in bilateral common peroneal nerves and absence of conduction in bilateral sural nerves. These symptoms resolved when further exposure to n-hexane was ceased but cold allodynia remained. Thus, cold allodynia and impaired temperature sense can be a manifestation of n-hexane neuropathy. Hence, abnormalities on nerve conduction studies can be detected in n-hexane neuropathy patients, even before clinical examination detects any such abnormalities. In the case of the patients presenting with sensory motor neuropathy, history of occupational exposure to n-hexane becomes important, as the sooner the disease is detected, the better the chances of recovery., (This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.)

Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.

Study on the impact of pollutants on cultural materials in storing or displaying micro-environment in museum is considered as very important for the preservation of cultural relics and its aging prevention. This paper applied the Fourier transform infrared (attenuated total reflection) technique to assess silk structural changes under volatile organic acids (formic acid/acetic acid), which usually come from decorative materials emission and commonly exist in the surface or around cultural materials. The focus of this work was on investigating the changes of peptide bond in the area of amide I-amide III, as well as the peptide chains (GlyAla), characteristic region. The structural and conformational changes in silk fiber treated with gaseous formic and acetic acid were assessed. The results indicate that both the gaseous acids can weaken the intermolecular hydrogen bond in fiber peptide, based on the spectral changes in the increased intensity of amide I (1 617 cm(-1)), the narrowing amide II peak (1 515 cm(-1)), the increased intensity of random coil conformation in amide III peak (1 230 cm(-1)), and the decreased fiber crystallinity as well. The obvious secondary structural conformation occurred when the concentration of gaseous formic acid reached 8.1 mg x m(-3) in simulated environment. The conformational transformation was supported by the observation of the rapidly reduced random coil conformation, the increased short peptide chains (GlyAla)n with beta-sheet conformation characteristic peak (1 000, 975 cm(-1)), and the enhanced fiber crystallinity degree as well. In contrast, gaseous acetic acid has less impact on the amide I and amide II bond based on the spectral changes, but it did promote random coil conformation and decreased fiber crystallinity. This work also provides a potential application of the infrared spectroscopy in non-destructive investigation of silk in-situ.

A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 μg or less of silk fiber. Except for a 2 h acid hydrolysis at 110 °C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids.

Synchrotron FTIR (S-FTIR) microspectroscopy was used to monitor the silk protein conformation in a range of single natural silk fibers (domestic and wild silkworm and spider dragline silk). With the selection of suitable aperture size, we obtained high-resolution S-FTIR spectra capable of semiquantitative analysis of protein secondary structures. For the first time, we have determined from S-FTIR the β-sheet content in a range of natural single silk fibers, 28 ± 4, 23 ± 2, and 17 ± 4% in Bombyx mori, Antheraea pernyi, and Nephila edulis silks, respectively. The trend of β-sheet content in different silk fibers from the current study accords quite well with published data determined by XRD, Raman, and (13)C NMR. Our results indicate that the S-FTIR microspectroscopy method has considerable potential for the study of single natural silk fibers.

Based on the phase diagram constructed for water-silk fibroin-LiBr using the osmotic stress method, wet-spinning of osmotically stressed, regenerated Bombyx mori silk fibroin was performed, without the necessity of using expensive or toxic organic solvents. The osmotic stress was applied to prestructure the regenerated silk fibroin molecule from its original random coil state to a more oriented state, manipulating the phase of the silk solution in the phase diagram before the start of spinning. Various starting points for spinning were selected from the phase diagram to evaluate the spinning performance and also physical properties of fibers produced. Monofilament fiber with a diameter of 20 microm was produced. It was found that the fibers whose starting point in the phase diagram were around the phase boundary between silk I and silk II, at very low LiBr concentrations, showed the best spinning process stability and physical properties. This underpins the prediction that the enhanced control over structure and phase behavior using the osmotic stress method helps improve the physical properties of wet-spun regenerated silk fibroin fibers.

Silks spun from the major (Ma) and minor (Mi) ampullate glands by the spider Nephila clavipes respond to water differently. Specifically, Ma silk supercontracts (shrinks 40-50% in length) while Mi silk does not contract at all when hydrated with water. In the present study, 1H --> 13C cross polarization magic angle spinning (CP-MAS), 13C MAS NMR collected with dipolar decoupling, and two-dimensional wide-line separation spectra are presented on Mi silk in its native and hydrated state and comparisons are made to Ma silk. This combination of NMR data demonstrates that water plasticizes Mi and Ma silk similarly, with an increase in chain dynamics observed in regions containing Gly, Glu, Ser, Tyr, Leu, and a fraction of Ala when the Mi silk is hydrated. Resonances that correspond to the poly(Ala) and poly(Gly Ala) motifs of Ma and Mi silk are predominately rigid indicating that water does not penetrate these beta-sheet domains.

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, (13)C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol-gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were alpha-helix and random coil in solution. There also existed a little beta-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no alpha-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol-gel transformation. The velocity increased obviously when the temperature was above 30 degrees C. During the sol-gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to beta-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.

Orb-web weaving spiders depend upon the mechanical performance of capture threads to absorb the energy of flying prey. Most orb-weavers spin wet capture threads with core fibers of flagelliform silk. These threads are extremely compliant and extensible due to the folding of their constituent proteins into molecular nanosprings and hydration by a surrounding coating of aqueous glue. In contrast, other orb-weavers use cribellate capture threads, which are composite structures consisting of core fibers of pseudoflagelliform silk surrounded by a matrix of fine dry cribellar fibrils. Based on phylogenetic evidence, cribellate capture threads predate the use of viscid capture threads. To better characterize how pseudoflagelliform and cribellar fibrils function, we investigated the mechanical performance of cribellate capture threads for three genera of spiders (Deinopis, Hyptiotes and Uloborus). These taxa spin very diverse web architectures, ranging from complete orbs to evolutionarily reduced triangle webs and cast nets. We found that the pseudoflagelliform core fibers of these webs were stiffer and stronger, but also less extensible, than flagelliform silk. However, cribellate capture threads achieved overall high extensibilities because the surrounding cribellar fibrils contributed substantially to the tensile performance of threads long after the core pseudoflagelliform fibers ruptured. In the case of Deinopis capture threads, up to 90% of the total work performed could be attributed to these fibrils. These findings yield insight into the evolutionary transition from cribellate to viscid capture threads.

There is evidence to suggest that Cu(II) is involved in the natural spinning process of a silkworm helping to convert the concentrated silk fibroin (SF) solution (or dope) into tough insoluble threads. To investigate the interaction between SF and Cu(II), a series of regenerated SF (RSF) films with different mass ratios of Cu(II) to SF were prepared. X-ray photoelectron spectroscopy (XPS) was employed to study the chemical interaction between Cu(II) and SF in these Cu(II)-RSF films. A significant change in the binding energy of Cu 2p(3/2) demonstrated that the chemical state of Cu(II) in the Cu(II)-RSF films was affected by the interaction between Cu(II) and SF. Moreover, chemical shifts of N 1s and O 1s of SF were also detected, implying that Cu(II) may coordinate with both N and O atoms in the SF. In addition, Raman spectra of the same series of Cu(II)-RSF films recorded the conformation transition of SF that may occur by the coordination of Cu(II) and SF macromolecular chains., (Copyright 2005 Wiley Periodicals, Inc.)

A simple solid-state NMR method was used to study the structure of (13)C- and (15)N-enriched silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. Carbon-13 and (15)N spectra from alanine- or glycine-labeled oriented dragline silks were acquired with the fiber axis aligned parallel or perpendicular to the magnetic field. The fraction of oriented component was determined from each amino acid, alanine and glycine, using each nucleus independently, and attributed to the ordered crystalline domains in the silk. The relative fraction of ordered alanine was found to be higher than the fraction of ordered glycine, akin to the observation of alanine-rich domains in silk-worm (Bombyx mori) silk. A higher degree of crystallinity was observed in the dragline silk of N. edulis compared with A. keyserlingi, which correlates with the superior mechanical properties of the former., (Copyright 2005 Wiley Periodicals, Inc.)

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.

Micrometer and submicrometer diameter fibers of recombinant dragline spider silk analogues, synthesized via protein engineering strategies, have been electrospun from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and compared with cast films via Raman spectroscopy in order to assess changes in protein conformation that may result from the electrospinning process. Although the solvent casting process was shown to result in predominantly beta-sheet conformation similar to that observed in the bulk, the electrospinning process causes a major change in conformation from beta-sheet to alpha-helix. A possible mechanism involving electric field-induced stabilization of alpha-helical segments in HFIP solution during the electrospinning process is discussed.

Spider silk is attractive because it is strong and tough. Moreover, an enormous range of mechanical properties can be achieved with only small changes in chemical structure. Our research shows that the full range of thermo-mechanical properties of silk fibres can be predicted from mean field theory for polymers in terms of chemical composition and the degree of order in the polymer structure. Thus, we can demonstrate an inherent simplicity at a macromolecular level in the design principles of natural materials. This surprising observation allows in depth comparison of natural with man-made materials.

Spiders have evolved to produce up to seven different silks; for each silk the balance between strength and elasticity is optimized according to the silk's specific use. Amino acid motifs have been identified and predicted functions have been assigned to these motifs. In addition to understanding the underlying molecular basis for structure/function relationships, it is also necessary to understand the contribution of the spinning process to the overall mechanical properties for both native and synthetic silk fibers. It is hypothesized that the natural silk fiber is produced through a process that involves dehydration. Current artificial spinning methods attempt to reproduce the natural dehydrating conditions by pulling the newly formed fiber through a methanol (or other dehydrating solvent) bath. However, the consequences of this treatment on the mechanical properties of the artificial fiber in relation to the natural fiber are unknown. To evaluate the effect of methanol on fiber properties, mechanical testing was done on native fibers from Nephila clavipes. Single major ampullate fibers were spun either in 47% ambient humidity or drawn post-spinning through a methanol bath. A comparison of the mechanical properties of the silk fibers revealed that post-spin drawing of a single major ampullate silk fiber through a methanol bath correlates to changes in the mechanical properties of the fiber.

Bombyx mori silk fibroin fiber is a fibrous protein produced by the silkworm at room temperature and from an aqueous solution whose primary structure is highly repetitive. In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using high-resolution solid-state 13C NMR. We establish that the Ser residue bearing a short polar side chain has the ability to stabilize the conformation formed in the model peptides due to its ability to form intermolecular hydrogen bonds involving its hydroxyl group as a donor and the carbonyl groups of other residues as acceptors. On the other hand, insertion of Tyr residue in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Moreover, the environmental effect was investigated by mixing the native Cp fraction with the model peptides, showing that there is no significant structural difference on the Ser-containing peptides, while structural transformation was observed on the peptides containing the GAAS unit. This may be attributed to the fact that the Cp fraction promotes the formation of an antiparallel beta-sheet in the Ala-Ala unit. Such periodically disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical not only for facilitating the conformational transformation from silk I to silk II structural form but also for having some correlation with the unique properties of the silk materials.