Seed-Specific Expression of Spider Silk Protein Multimers Causes Long-Term Stability.
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ABSTRACT:
SUBMITTER: Weichert N
PROVIDER: S-EPMC4729946 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The emergence of order from disorder is a topic of vital interest. We here propose that long-range order can arise from a randomly arranged two-phase material under mechanical load. Using Small-Angle Neutron Scattering (SANS) experiments and Molecular Dynamics based finite element (FE) models we show evidence for stress-induced ordering in spider dragline silk. Both methods show striking quantitative agreement of the position, shift and intensity increase of the long period upon stretching. We demonstrate that mesoscopic ordering does not originate from silk-specific processes such as strain-induced crystallization on the atomistic scale or the alignment of tilted crystallites. It instead is a general phenomenon arising from a non-affine deformation that enhances density fluctuations of the stiff and soft phases along the direction of stress. Our results suggest long-range ordering, analogously to the coalescence of defects in materials, as a wide-spread phenomenon to be exploited for tuning the mechanical properties of many hybrid stiff and soft materials.
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Project description:Biotechnological production is a powerful tool to design materials with customized properties. The aim of this work was to apply designed spider silk proteins to produce Janus fibers with two different functional sides. First, functionalization was established through a cysteine-modified silk protein, ntagCys eADF4(κ16). After fiber spinning, gold nanoparticles (AuNPs) were coupled via thiol-ene click chemistry. Significantly reduced electrical resistivity indicated sufficient loading density of AuNPs on such fiber surfaces. Then, Janus fibers were electrospun in a side-by-side arrangement, with "non-functional" eADF4(C16) on the one and "functional" ntagCys eADF4(κ16) on the other side. Post-treatment was established to render silk fibers insoluble in water. Subsequent AuNP binding was highly selective on the ntagCys eADF4(κ16) side demonstrating the potential of such silk-based systems to realize complex bifunctional structures with spatial resolutions in the nano scale.
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Project description:Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering.
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Project description:The present study demonstrates pDNA complexes of recombinant silk proteins containing poly(L-lysine) and tumor-homing peptides (THPs), which are globular and approximately 150-250 nm in diameter, show significant enhancement of target specificity to tumor cells by additions of F3 and CGKRK THPs. We report herein the preparation and study of novel nanoscale silk-based ionic complexes containing pDNA able to home specifically to tumor cells. Particular focus was on how the THP, F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), and CGKRK, enhanced transfection specificity to tumor cells. Genetically engineered silk proteins containing both poly(L-lysine) domains to interact with pDNA and the THP to bind to specific tumor cells for target-specific pDNA delivery were prepared using Escherichia coli, followed by in vitro and in vivo transfection experiments into MDA-MB-435 melanoma cells and highly metastatic human breast tumor MDA-MB-231 cells. Non-tumorigenic MCF-10A breast epithelial cells were used as a control cell line for in vitro tumor-specific delivery studies. These results demonstrate that combination of the bioengineered silk delivery systems and THP can serve as a versatile and useful new platform for nonviral gene delivery.
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Project description:BACKGROUND: It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. METHODOLOGY/PRINCIPAL FINDINGS: We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. CONCLUSIONS: Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics.
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Project description:Spider dragline silk has unique characteristics of strength and extensibility, including supercontraction. When we use it as a biomaterial or material for textiles, it is important to suppress the effect of water on the fiber by as much as possible in order to maintain dimensional stability. In order to produce spider silk with a highly hydrophobic character, based on the sequence of ADF-3 silk, we produced recombinant silk (RSSP(VLI)) where all QQ sequences were replaced by VL, while single Q was replaced by I. The artificial RSSP(VLI) fiber was prepared using formic acid as the spinning solvent and methanol as the coagulant solvent. The dimensional stability and water absorption experiments of the fiber were performed for eight kinds of silk fiber. RSSP(VLI) fiber showed high dimensional stability, which is suitable for textiles. A remarkable decrease in the motion of the fiber in water was made evident by 13C solid-state NMR. This study using 13C solid-state NMR is the first trial to put spider silk to practical use and provide information regarding the molecular design of new recombinant spider silk materials with high dimensional stability in water, allowing recombinant spider silk proteins to be used in next-generation biomaterials and materials for textiles.
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Project description:High-performance functional biomaterials are becoming increasingly requested. Numerous natural and artificial polymers have already demonstrated their ability to serve as a basis for bio-composites. Spider silk offers a unique combination of desirable aspects such as biocompatibility, extraordinary mechanical properties, and tunable biodegradability, which are superior to those of most natural and engineered materials. Modifying spider silk with various inorganic nanomaterials with specific properties has led to the development of the hybrid materials with improved functionality. The purpose of using these inorganic nanomaterials is primarily due to their chemical nature, enhanced by large surface areas and quantum size phenomena. Functional properties of nanoparticles can be implemented to macro-scale components to produce silk-based hybrid materials, while spider silk fibers can serve as a matrix to combine the benefits of the functional components. Therefore, it is not surprising that hybrid materials based on spider silk and inorganic nanomaterials are considered extremely promising for potentially attractive applications in various fields, from optics and photonics to tissue regeneration. This review summarizes and discusses evidence of the use of various kinds of inorganic compounds in spider silk modification intended for a multitude of applications. It also provides an insight into approaches for obtaining hybrid silk-based materials via 3D printing.
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Project description:Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of ?-sheets to lock in structures via physical interactions without the need for chemical cross-linking. These new functional silk proteins were assessed for antimicrobial activity against Gram - Escherichia coli and Gram + Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.
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Project description:The ultimate aim of flow sensing is to represent the perturbations of the medium perfectly. Hundreds of millions of years of evolution resulted in hair-based flow sensors in terrestrial arthropods that stand out among the most sensitive biological sensors known, even better than photoreceptors which can detect a single photon (10-18-10-19 J) of visible light. These tiny sensory hairs can move with a velocity close to that of the surrounding air at frequencies near their mechanical resonance, despite the low viscosity and low density of air. No man-made technology to date demonstrates comparable efficiency. Here we show that nanodimensional spider silk captures fluctuating airflow with maximum physical efficiency (Vsilk/Vair ? 1) from 1 Hz to 50 kHz, providing an effective means for miniaturized flow sensing. Our mathematical model shows excellent agreement with experimental results for silk with various diameters: 500 nm, 1.6 µm, and 3 µm. When a fiber is sufficiently thin, it can move with the medium flow perfectly due to the domination of forces applied to it by the medium over those associated with its mechanical properties. These results suggest that the aerodynamic property of silk can provide an airborne acoustic signal to a spider directly, in addition to the well-known substrate-borne information. By modifying a spider silk to be conductive and transducing its motion using electromagnetic induction, we demonstrate a miniature, directional, broadband, passive, low-cost approach to detect airflow with full fidelity over a frequency bandwidth that easily spans the full range of human hearing, as well as that of many other mammals.
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Project description:Spider silk is frequently attributed antimicrobial properties. This notion is based on studies reporting antimicrobial activity (AMA) of spider silk; however, close inspection of these studies reveals that the evidence is conflicting, and at best anecdotal. We performed a systematic study of antimicrobial properties of different silk types from seven species across the spider phylogeny. We found no evidence of AMA of silk in direct contact and disc diffusion assays against Gram-negative Escherichia coli and Pseudomonas putida, and the Gram-positive Bacillus subtilis. Furthermore, staining experiments and fluorescence microscopy showed the presence of live bacteria on silk surfaces indicating no antimicrobial effect on direct contact. A critical evaluation of the literature reveals that published tests of AMA are scarce and that all the studies claiming positive results are compromised by methodological shortcomings. Our analysis demonstrates that the common notion that spider silk is antimicrobial is not supported by empirical data.
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