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1

Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts.  

PubMed

The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta cocoon has been assessed and compared with that of the mulberry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive control for the experiment. Our results showed that the sericin obtained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-incubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase; lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant. PMID:18377728

Dash, Rupesh; Acharya, Chitrangada; Bindu, P C; Kundu, S C

2008-03-31

2

Silk gland sericin protein membranes: fabrication and characterization for potential biotechnological applications.  

PubMed

This study describes the potential use of silk gland sericin protein as a biocompatible natural biopolymer in its native form. The membranes were fabricated using native silk sericin protein extracted from middle silk gland of Antheraea mylitta, a non-mulberry tropical tasar silkworm without using any cross-linking agent. The fabricated membranes were biophysically characterized and optimized for cell culture. Silk sericin protein extracted from gland contained higher amount of beta-sheets, which increased upon treatment with ethanol as observed by FTIR and XRD. The membranes did show robustness, good mechanical strength and high temperature stability. Cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis using feline fibroblast cells. Morphology of growing cells was assessed by confocal microscopy that indicated normal spreading and proliferation on the silk sericin membranes. The membranes showed low inflammatory response as observed assaying TNF alpha release. This study reveals the potential of native silk sericin protein from silk gland as biocompatible biopolymer for potential biomedical applications. PMID:19808068

Dash, Biraja C; Mandal, Biman B; Kundu, S C

2009-10-04

3

Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties.  

PubMed

In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibroin structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 degrees C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS-PAGE), respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS-PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 degrees C. PMID:20460689

Teh, Thomas K H; Toh, Siew-Lok; Goh, James C H

2010-05-11

4

An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms.  

PubMed

The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins. PMID:23435751

Wang, Feng; Xu, Hanfu; Yuan, Lin; Ma, Sanyuan; Wang, Yuancheng; Duan, Xiaoli; Duan, Jianping; Xiang, Zhonghuai; Xia, Qingyou

2013-02-23

5

Separation of sericin from fatty acids towards its recovery from silk degumming wastewaters  

Microsoft Academic Search

Sericin is a valuable protein, which is currently discarded as a waste in silk industry. Silk degumming wastewaters (SDW) are abundant sources of sericin, however, they contain impurities such as salts of fatty acids (soap) in conventional degumming processes. In this study, a process consisting of centrifugation (CFG), low temperature crystallization (LTC) and ultrafiltration (UF) was developed to separate sericin

Goksen Capar; S. Seylan Aygun; M. Rusen Gecit

2009-01-01

6

Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing.  

PubMed

Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable beta-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing. PMID:19060395

Teramoto, Hidetoshi; Kameda, Tsunenori; Tamada, Yasushi

2008-12-07

7

Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta.  

PubMed

A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8M urea containing 1% sodium dodecyl sulfate and beta-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified by gel filtration chromatography. In SDS-PAGE, a single band of approximately 200kDa was detected both in non-reducing and reducing conditions. Amino acid analysis showed that the protein is enriched in glycine and serine. There is a slight difference observed in amino acid composition between the sericin from cocoon peduncle and cocoon of A. mylitta. Secondary structure estimation by circular dichroism spectrometry showed 36.7% beta-sheets, 52.7% random coils, 10.6% turns and no helices. PMID:16620954

Dash, Rupesh; Mukherjee, Soumen; Kundu, S C

2006-03-07

8

Non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices for potential biotechnological applications.  

PubMed

This study reports a novel biopolymeric matrix fabricated by chemically cross-linking poly (vinyl alcohol) with silk sericin protein obtained from cocoons of the tropical tasar silkworm Antheraea mylitta. Glutaraldehyde was used as a cross-linking agent with hydrochloric acid acting as an initiator. The matrices were biophysically characterized and the cytocompatibility of the matrices was evaluated for their suitability as biomaterials. The surface morphology was assessed using atomic force microscopy while the changes taking place after cross-linking were confirmed by Fourier transform infrared spectroscopy. The enhanced thermal stability of the constructs was assessed by thermogravimetric and differential scanning calorimetry. Fourier transform infrared spectroscopy analysis showed that sericin was chemically cross-linked with poly (vinyl alcohol) using glutaraldehyde. Silk sericin protein demonstrated a favorable effect on animal cell culture by successfully improving the adhering and spreading of cells on the poorly adhering surface of poly (vinyl alcohol). Confocal microscopy revealed cell spreading and actin filament development in sericin/poly (vinyl alcohol) hydrogel matrices. These findings prove the potential of non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices to be used as biocompatible and biopolymeric material for tissue-engineering and biotechnological applications. PMID:21549749

Mandal, Biman B; Ghosh, Borna; Kundu, S C

2011-04-28

9

Dietary silk protein, sericin, improves epidermal hydration with increased levels of filaggrins and free amino acids in NC/Nga mice.  

PubMed

Epidermal hydration is maintained primarily by natural moisturising factors (NMF), of which free amino acids (AA) are major constituents that are generated by filaggrin degradation. To identify dietary sources that may improve skin dryness of atopic dermatitis (AD), we investigated dietary effects of silk proteins, sericin and fibroin, on epidermal levels of hydration, filaggrins and free AA, as well as PPAR?, peptidylarginine deiminase-3 (PAD3) and caspase-14 proteins involved in filaggrin expression and degradation processes. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or diets with 1 % sericin (group S) or fibroin (group F) for 10 weeks. In group S, epidermal levels of hydration, total filaggrins and total free AA, as well as PPAR?, PAD3 and caspase-14, which were reduced in group CA, were increased to higher or similar levels of a normal control group of BALB/c mice (group C). Furthermore, profilaggrin, a precursor with multiple filaggrin repeats, and three repeat intermediates were increased, while two repeat intermediates and filaggrin were decreased in parallel with increased levels of glutamate and serine, major AA of NMF in group S. Despite increased levels of total filaggrins, total free AA, PPAR? and PAD3, epidermal levels of hydration, glutamate, serine and caspase-14 were not increased, but other minor AA of NMF were highly detected in group F. Dietary sericin improves epidermal hydration in parallel with enhancing profilaggrin expression and degradation into free AA that is coupled with elevated levels of PPAR?, PAD3 and caspase-14 proteins. PMID:22244094

Kim, Hyunae; Lim, Yu-ji; Park, Ji-Ho; Cho, Yunhi

2012-01-16

10

The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds  

Microsoft Academic Search

Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation\\u000a may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming\\u000a silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin\\u000a tissue engineering. The objective

Tippawan Siritientong; Teerapol Srichana; Pornanong Aramwit

2011-01-01

11

Cloning, expression, and assembly of sericin-like protein.  

PubMed

Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber. PMID:12963711

Huang, Jia; Valluzzi, Regina; Bini, Elisabetta; Vernaglia, Brian; Kaplan, David L

2003-09-08

12

Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.  

PubMed

Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation. PMID:22860103

Martínez-Mora, Celia; Mrowiec, Anna; García-Vizcaíno, Eva María; Alcaraz, Antonia; Cenis, José Luis; Nicolás, Francisco José

2012-07-31

13

Fibroin and Sericin from Bombyx mori Silk Stimulate Cell Migration through Upregulation and Phosphorylation of c-Jun  

PubMed Central

Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation.

Garcia-Vizcaino, Eva Maria; Alcaraz, Antonia; Cenis, Jose Luis; Nicolas, Francisco Jose

2012-01-01

14

Mineralization and Biocompatibility of Antheraea pernyi (A. pernyi) Silk Sericin Film for Potential Bone Tissue Engineering.  

PubMed

This study aimed to investigate the mineralization of Antheraea pernyi (A. pernyi) silk sericin. Mineralization of A. pernyi sericin was performed by alternative soaking in calcium and phosphate. The inhibition of precipitation of calcium carbonate and von Kossa staining on A. pernyi sericin were tested, and the corresponding results prove that A. pernyi sericin has Ca binding activity. Scanning electron microscope (SEM) observation shows that spherical crystals could be nucleated on the A. pernyi sericin film. These crystals were confirmed to be hydroxyapatite according to FT-IR and XRD spectra, indicating that A. pernyi sericin is capable of mineralization. In addition, cell adhesion and growth activity assay demonstrate that A. pernyi sericin shows excellent biocompatibility for the growth of MG-63 cells. PMID:24092210

Yang, Mingying; Mandal, Namita; Shuai, Yajun; Zhou, Guanshan; Min, Sijia; Zhu, Liangjun

2013-01-01

15

Silk sericin/polyacrylamide in situ forming hydrogels for dermal reconstruction.  

PubMed

In situ forming tissue sealants are advantageous due to ease in application, complete coverage of defect site and assured comfort levels to patients. The interconnected three-dimensional hydrophilic networks perfectly manage typical dermal wounds by suitably scaffolding skin fibroblast, diffusing the nutrients, therapeutics and exudates while still maintaining an adequately moist environment. We evaluate the cell homing ability of semi-interpenetrating non-mulberry tropical tasar silk sericin/polyacrylamide hydrophilic network with a keen understanding of its network characteristics and correlation of protein concentration with the performance as cell scaffold. Interconnectivity of porous networks observed through scanning electron micrograph revealed pore sizes ranging from 23 to 52 ?m. The enhanced ?-sheet content with the increasing sericin concentration in far red spectroscopy study supported their corresponding improved compressive strength. These semi-interpenetrating networks were found to possess a maximum fluid uptake of 112% of its weight, hence preventing the accumulation of exudates at the wound area. The present systems appear to possess characteristics like rapid gelation (~5min) at 37 °C, 98% porosity enabling the migration of fibroblasts during healing (observed through confocal and scanning electron micrographs), cell adhesion together with the absence of any cyto-toxic effect suggesting its potential as in situ tissue sealants. The compressive strength up to 61 kPa ensured ease in handling even when wet. The results prove the suitability to use non-mulberry tasar cocoon silk sericin/polyacrylamide semi-interpenetrating network as a reconstructive dermal sealant. PMID:22819495

Kundu, Banani; Kundu, Subhas C

2012-07-21

16

Study on silk sericin and chitosan blend film: morphology and secondary structure characterizations.  

PubMed

This study aimed to prepare and characterize silk sericin and chitosan blend film as well as the native silk sericin and chitosan films. The films were observed their morphology using Scanning Electron Microscope (SEM). The secondary structures of the films were analyzed using Fourier Transform Infrared (FTIR) spectroscopy. Transparency of the films was investigated with UV-visible spectroscopy. The results found that all of silk films were smooth throughout the film surfaces, including blend film. This showed that silk sericin and chitosan very well compatible. However, phase separation is also being observed. It is show that the interaction between two materials might be miscible together. The FTIR results indicated that the most of films were composed both in random coil and beta-sheet forms which predominantly of the random coil structures. The results suggesting the blend film between sericin and chitosan did not change the intramolecular structure when compared to the native films. The silk sericin and blend films were slightly yellowish color and were higher transparent than chitosan film. However, % transmittance at lamda max of 660 nm showed that all of films have similar values. The result suggested that the transparency of the film did not change even blend together. It is a promising that both silk sericin and chitosan would be blended into many forms for applications in specifically fields. PMID:20180324

Srihanam, P; Simcheur, W; Srisuwan, Y

2009-11-15

17

Novel silk sericin/gelatin 3-D scaffolds and 2-D films: fabrication and characterization for potential tissue engineering applications.  

PubMed

In this study, we report for the first time the fabrication of novel 3-D sericin/gelatin scaffolds and 2-D films using non-mulberry Antheraea mylitta silk cocoon sericin protein. The matrices were fabricated, biophysically characterized and optimized for cell culture applications. Blended sericin/gelatin 3-D scaffolds were highly porous with an optimum pore size of 170+/-20 microm. The scaffolds were robust with enhanced mechanical strength and showed high compressibility. Swelling studies showed high swellability along with complete degradation in the presence of phosphate-buffered saline. Cytocompatibility of the matrices was evaluated using feline fibroblasts showing normal spreading and proliferation as assessed by fluorescence microscopy. Cell cycle analysis showed cytocompatibility without any cell cycle arrests. Low immunogenicity of the matrices as observed through tumor necrosis factor alpha release reveal its potential as future biopolymeric graft material. The results of this novel study lay the foundation for the use of the silk cocoon protein sericin as a biocompatible biopolymer for tissue engineering applications. PMID:19398392

Mandal, Biman B; Priya, Anjana S; Kundu, S C

2009-03-31

18

Bioconjugation of neutral protease on silk fibroin nanoparticles and application in the controllable hydrolysis of sericin.  

PubMed

Bombyx mori silk fibroin is a protein-based macromolecular biopolymer with remarkable biocompatibility. Silk fiber was degummed and subjected to a series of treatments, including dissolution and dialysis, to yield an aqueous solution of silk fibroin, which was introduced rapidly into excess acetone to produce crystalline silk fibroin nanoparticles (SFNs). The SFNs were conjugated covalently with a neutral protease (NP) using glutaraldehyde as the cross-linking reagent. The objective of this study was to determine the optimal conditions for biosynthesis of the SFN-NP bioconjugates. First, SFN-NP was obtained by covalent cross-linking of SFN and NP at an SFN/NP ratio of 5-8 mg:1 IU with 0.75% glutaraldehyde for 6 h at 25 °C. When adding 50 IU of the enzyme, the residual activity of biological conjugates was increased to 31.45%. Studies on the enzyme activity of SFN-NP and its kinetics showed that the stability of SFN-NP bioconjugates was greater than that of the free enzyme, the optimum reactive temperature range was increased by 5-10 °C, and the optimum pH value range was increased to 6.5-8.0. Furthermore, the thermal stability was improved to some extent. A controlled hydrolysis test using the poorly water-soluble protein sericin as a substrate and SFN-NP as the enzyme showed that the longer the reaction time (within 1 h), the smaller the molecular mass (<30 kDa) is of the sericin peptide produced. The SFN-NP bioconjugate is easily recovered by centrifugation and can be used repeatedly. The highly efficient processing technology and the use of SFN as a novel vector for a protease has great potential for research and the development of food processing. PMID:21846144

Zhu, Lin; Hu, Ren-Ping; Wang, Hai-Yan; Wang, Yuan-Jing; Zhang, Yu-Qing

2011-08-25

19

Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery  

NASA Astrophysics Data System (ADS)

In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites.

Mandal, Biman B.; Kundu, S. C.

2009-09-01

20

Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery.  

PubMed

In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites. PMID:19671963

Mandal, Biman B; Kundu, S C

2009-08-11

21

Preliminary Characterization of Genipin-Cross-Linked Silk Sericin/Poly(vinyl alcohol) Films as Two-Dimensional Wound Dressings for the Healing of Superficial Wounds  

PubMed Central

The genipin-cross-linked silk sericin/poly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1%?w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds.

Siritientong, Tippawan; Ratanavaraporn, Juthamas; Srichana, Teerapol; Aramwit, Pornanong

2013-01-01

22

Bioengineered silk proteins to control cell and tissue functions.  

PubMed

Silks are defined as protein polymers that are spun into fibers by some lepidoptera larvae such as silkworms, spiders, scorpions, mites, and flies. Silk proteins are usually produced within specialized glands in these animals after biosynthesis in epithelial cells that line the glands, followed by secretion into the lumen of the gland prior to spinning into fibers.The most comprehensively characterized silks are from the domesticated silkworm (Bombyx mori) and from some spiders (Nephila clavipes and Araneus diadematus). Silkworm silk has been used commercially as biomedical sutures for decades and in textile production for centuries. Because of their impressive mechanical properties, silk proteins provide an important set of material options in the fields of controlled drug release, and for biomaterials and scaffolds for tissue engineering. Silkworm silk from B. mori consists primarily of two protein components, fibroin, the structural protein of silk fibers, and sericins, the water-soluble glue-like proteins that bind the fibroin fibers together. Silk fibroin consists of heavy and light chain polypeptides linked by a disulfide bond. Fibroin is the protein of interest for biomedical materials and it has to be purified/extracted from the silkworm cocoon by removal of the sericin. Characteristics of silks, including biodegradability, biocompatibility, controllable degradation rates, and versatility to generate different material formats from gels to fibers and sponges, have attracted interest in the field of biomaterials. Cell culture and tissue formation using silk-based biomaterials have been pursued, where appropriate cell adhesion, proliferation, and differentiation on or in silk biomaterials support the regeneration of tissues. The relative ease with which silk proteins can be processed into a variety of material morphologies, versatile chemical functionalization options, processing in water or solvent, and the related biological features of biocompatibility and enzymatic degradability make these proteins interesting candidates for biomedical applications. PMID:23504416

Preda, Rucsanda C; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L

2013-01-01

23

Comparative Proteomics Reveal Diverse Functions and Dynamic Changes of Bombyx mori Silk Proteins Spun from Different Development Stages.  

PubMed

Silkworms (Bombyx mori) produce massive amounts of silk proteins to make cocoons during the final stages of larval development. Although the major components, fibroin and sericin, have been the focus for a long time, few researchers have realized the complexity of the silk proteome. We collected seven kinds of silk fibers spun by silkworm larvae at different developmental stages: the silks spun by new hatched larvae, second instar day 0 larvae, third instar day 0 larvae, fourth instar day 0 larvae, and fourth instar molting larvae, the scaffold silk used to attach the cocoon to the substrate and the cocoon silk. Analysis by liquid chromatography-tandem mass spectrometry identified 500 proteins from the seven silks. In addition to the expected fibroins, sericins, and some known protease inhibitors, we also identified further protease inhibitors, enzymes, proteins of unknown function, and other proteins. Unsurprisingly, our quantitative results showed fibroins and sericins were the most abundant proteins in all seven silks. Except for fibroins and sericins, protease inhibitors, enzymes, and proteins of unknown function were more abundant than other proteins. We found significant change in silk protein compositions through development, being consistent with their different biological functions and complicated formation. PMID:24093152

Dong, Zhaoming; Zhao, Ping; Wang, Chen; Zhang, Yan; Chen, Jianping; Wang, Xin; Lin, Ying; Xia, Qingyou

2013-10-08

24

The influence of silkworm species on cellular interactions with novel PVA/silk sericin hydrogels.  

PubMed

Sericin peptides and PVA are chemically modified with methacrylate groups to produce a covalent PVA/sericin hydrogel. Preservation of the sericin bioactivity following methacrylation is confirmed, and PVA/sericin hydrogels are fabricated for both B. mori and A. mylitta sericin. Cell adhesion studies confirm the preservation of sericin bioactivity post incorporation in PVA gels. PVA/A. mylitta gels are observed to facilitate cell adhesion to a significantly greater degree than PVA/B. mori gels. Overall, the incorporation of sericin does not alter the physical properties of the PVA hydrogels but does result in significantly improved cellular interaction, particularly from A. mylitta gels. PMID:22493796

Lim, Khoon S; Kundu, Joydip; Reeves, April; Poole-Warren, Laura A; Kundu, Subhas C; Martens, Penny J

2012-03-01

25

Study of the physical properties of whey protein: sericin protein-blended edible films  

Microsoft Academic Search

Effects of the sericin protein on the physical properties of the whey protein isolate film including the mechanical strength,\\u000a water vapor permeability, light transmission, moisture content, solubility and swelling were investigated in the present work.\\u000a The mechanical strength of the whey protein film could be reinforced with 0.1% sericin caused by the hydrogen bonding between\\u000a the sericin and whey protein

Jing Wang; Jiejing Shang; Fazheng Ren; Xiaojing Leng

2010-01-01

26

Production and properties of electrosprayed sericin nanopowder  

NASA Astrophysics Data System (ADS)

Sericin is a proteinous substrate that envelops fibroin (silk) fiber, and its recovery provides significant economical and social benefits. Sericin is an antibacterial agent that resists oxidation and absorbs moisture and UV light. In powder form, sericin has a wide range of applications in food, cosmetics and drug delivery. Asides from other techniques of producing powder, such as precipitation and spray drying, electrospraying can yield solid nanoparticles, particularly in the submicron range. Here, we report the production of sericin nanopowder by electrospraying. Sericin sponge was recovered from Bombyx mori cocoons through a high-temperature, high-pressure process, followed by centrifugation and freeze drying of the sericin solution. The electrospraying solution was prepared by dissolving the sericin sponge in dimethyl sulfoxide. We demonstrate that electrospraying is capable of producing sericin nanopowder with an average particle size of 25 nm, which is by far smaller than the particles produced by other techniques. The electrosprayed sericin nanopowder consists of small crystallites and exhibits a high moisture absorbance.

Hazeri, Najmeh; Tavanai, Hossein; Moradi, Ali Reza

2012-06-01

27

Preliminary Characterization of Genipin-Cross-Linked Silk Sericin/Poly(vinyl alcohol) Films as Two-Dimensional Wound Dressings for the Healing of Superficial Wounds.  

PubMed

The genipin-cross-linked silk sericin/poly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1%?w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds. PMID:24106722

Siritientong, Tippawan; Ratanavaraporn, Juthamas; Srichana, Teerapol; Aramwit, Pornanong

2013-09-11

28

Silk-based biomaterials  

Microsoft Academic Search

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined

Gregory H. Altman; Frank Diaz; Caroline Jakuba; Tara Calabro; Rebecca L. Horan; Jingsong Chen; Helen Lu; John Richmond; David L. Kaplan

2003-01-01

29

Potential of 2D crosslinked sericin membranes with improved biostability for skin tissue engineering.  

PubMed

Silk sericin protein is a natural, hydrophilic, macromolecular glycoprotein mainly synthesized in the middle silk gland of the silkworm. It constitutes 25-30% of the silk cocoon. Sericin proteins have antioxidant, antimicrobial, UV-resistant properties, promote wound healing and support cell proliferation even in serum-free media. Most of the sericin is discarded as waste in silk processing industries. This study aims at improving the mechanical strength and stability of sericin extracted from the silk cocoons during processing and utilize it as a biocompatible natural biopolymer in biomedical applications. Crosslinked sericin membranes, from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, were prepared using gluteraldehyde as the crosslinking agent. Physical and structural characteristics of the membranes were analyzed using scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and X-ray diffraction along with swelling and degradation studies. The secondary structure of the membrane indicates that crosslinking provides a more integrated structure that significantly improves the stability and mechanical strength of the membranes. In vitro cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis of feline fibroblast cells. The adherence, growth and proliferation patterns of cells on membranes were assessed by confocal microscopy, which demonstrated that the latter is non-toxic and supports cell growth. Cell cycle analyses indicate cytocompatibility with normal cell cycle pattern. This study reveals that silk sericin protein can be used as a biocompatible natural biopolymer for various applications in the biomedical field. PMID:22327482

Nayak, Sunita; Talukdar, Sarmistha; Kundu, Subhas C

2012-02-12

30

Dietary sericin enhances epidermal levels of glucosylceramides and ceramides with up-regulating protein expressions of glucosylceramide synthase, ?-glucocerebrosidase and acidic sphingomyelinase in NC/Nga mice.  

PubMed

We have previously reported that dietary sericin improves epidermal dryness with the increased total Ceramide (Cer) in NC/Nga mice, an animal model of atopic dermatitis (AD). In this study, we hypothesized that the increased level of total Cer induced by dietary sericin would be related to the altered metabolism of glucosylceramide (GlcCer) and sphingomyelin (SM), major precursors of Cer generation. NC/Nga mice were fed a control diet (group CA: atopic control) or diets with 1% silk protein, either sericin (group S) or fibroin (group F) for 10 weeks. In the epidermis of group CA, total Cer (including Cer1, 2, 3/4 and 6) and all GlcCer species were reduced; these levels in group S were increased to levels similar to or higher than in the normal control group of BALB/c mice (group C). In addition, the protein expressions, but not mRNA expressions, of GlcCer synthase, ?-glucocerebrosidase, and acidic sphingomyelinase, enzymes for GlcCer synthesis, GlcCer and SM hydrolysis, respectively, were highly increased in group S. The epidermal levels of total Cer (including Cer2, 3/4, and 6) and all GlcCer species and of these enzyme proteins in group F were lower than in group S. Notably, alterations in total SM, SM1, SM3, and SM synthase 1, which were increased in group CA, were not significant between groups S and F. Cer5 and SM2 were not altered among groups. Dietary sericin enhanced the epidermal levels of all GlcCer and most Cer species with up-regulating protein expressions of GlcCer synthase, ?-glucocerebrosidase, and acidic sphingomyelinase. PMID:23244541

Kim, Hyunae; Lee, Jongsun; Cho, Yunhi

2012-10-23

31

Recombinant DNA production of spider silk proteins.  

PubMed

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

2013-11-01

32

Bioprocessing of silk proteins-controlling assembly  

Microsoft Academic Search

A model for silk processing in silkworms and spiders is proposed and based on the unique domain structure in the sequences of silk proteins. A hypothesis for the silk-spinning process begins with chain folding at lower concentrations of protein, proceeds through formation of micelles and then micellar aggregates (globules) through water loss and increasing protein concentration, and finally leads to

Hyoung-Joon Jin; Jaehyung Park; Regina Valluzi; Ung-Jin Kim; Peggy Cebe; David Kaplan

33

Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta.  

PubMed

Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons). PMID:17350301

Dash, Rupesh; Ghosh, Sudip K; Kaplan, David L; Kundu, S C

2007-01-30

34

Single Honeybee Silk Protein Mimics Properties of Multi-Protein Silk  

PubMed Central

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.

Sutherland, Tara D.; Church, Jeffrey S.; Hu, Xiao; Huson, Mickey G.; Kaplan, David L.; Weisman, Sarah

2011-01-01

35

Sequence Requirements of Spider Silk Elastic Proteins.  

National Technical Information Service (NTIS)

Sequence for flagelliform silk proteins from Nephila, Argiope, and Araneus species have been obtained. These sequences show significant sequence differences although the changes are not predicted to affect the secondary structure of the protein and theref...

R. V. Lewis

2001-01-01

36

Consumption of a resistant protein, sericin, elevates fecal immunoglobulin A, mucins, and cecal organic acids in rats fed a high-fat diet.  

PubMed

We previously reported that consumption of a resistant protein, sericin, reduces colon tumorigenesis, constipation, and serum TG in rodents. The present study was conducted to elucidate the effects of dietary sericin on the intestinal luminal environment in rats fed a high-fat (HF) diet. Rats were fed 300 or 50 g/kg of beef tallow with or without 40 g/kg sericin, a protein purified from cocoons of Bombix mori, for 3 wk. Intestinal luminal variables, including IgA (index of intestinal immune function), mucins (index of barrier function), organic acids, microflora, and secondary bile acids, were measured. Dietary sericin markedly elevated fecal IgA in the HF diet group (3-fold, P < 0.05) but not in the low-fat (LF) diet group. Fecal mucin levels were elevated by sericin intake in the HF diet group (P < 0.05). Cecal organic acids, including acetate, propionate, n-butyrate, and succinate, were significantly lower in the HF diet group compared with the LF diet group. Dietary sericin significantly elevated cecal acetate and n-butyrate in the HF diet group but not in the LF diet group. Compared with the LF diet, the HF diet significantly increased serum TG in the untreated group but not in those fed sericin. The HF diet increased lower density lipoprotein (VLDL + IDL + LDL) cholesterol and it was reduced by sericin intake (P < 0.05). There was an inverse correlation between serum TG and cecal acetate (Spearman rank correlation coefficient = -0.63; P < 0.001). The profile of microflora in cecal digesta and fecal secondary bile acids (a risk factor for colon cancer) did not differ between the HF diet and HF diet with sericin groups. These results suggest a novel and favorable effect of sericin on colon health by modulating intestinal immune and barrier functions and fermentation in rats fed a HF diet. PMID:21940508

Okazaki, Yukako; Tomotake, Hiroyuki; Tsujimoto, Kazuhisa; Sasaki, Masahiro; Kato, Norihisa

2011-09-21

37

Effect of Processing on Silk-Based Biomaterials: Reproducibility and Biocompatibility  

PubMed Central

Silk fibroin has been successfully used as a biomaterial for tissue regeneration. In order to prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or ?-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0% and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes.

Wray, Lindsay S.; Hu, Xiao; Gallego, Jabier; Georgakoudi, Irene; Omenetto, Fiorenzo G.; Schmidt, Daniel; Kaplan, David L.

2012-01-01

38

Elastic theory of single spider silk protein molecule  

Microsoft Academic Search

Spider silk exhibits two excellent mechanical features, toughness and extensibility. In recent years, considerable investigations have been focused on it. The understanding of spider silk protein is significant for the bionic silk. In this article, we study the force extension property of spider silk proteins as the function of the amino acid compositions for major and minor ampullate glands of

Luru Dai; Yong Zhang; Zhong-can Ou-Yang

2003-01-01

39

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons.  

PubMed

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori. PMID:19633923

Wen, Hongxiu; Lan, Xiqian; Zhang, Yuansong; Zhao, Tianfu; Wang, Yujun; Kajiura, Zenta; Nakagaki, Masao

2009-07-25

40

Structure of a Protein Superfiber: Spider Dragline Silk  

Microsoft Academic Search

Spider major ampullate (dragline) silk is an extracellular fibrous protein with unique characteristics of strength and elasticity. The silk fiber has been proposed to consist of pseudocrystalline regions of antiparallel beta-sheet interspersed with elastic amorphous segments. The repetitive sequence of a fibroin protein from major ampullate silk of the spider Nephila clavipes was determined from a partial cDNA clone. The

Ming Xu; Randolph V. Lewis

1990-01-01

41

Recombinant production of spider silk proteins.  

PubMed

Natural spider silk fibers combine extraordinary properties such as stability and flexibility which results in a toughness superseding that of all other fiber materials. As the spider's aggressive territorial behavior renders their farming not feasible, the biotechnological production of spider silk proteins (spidroins) is essential in order to investigate and employ them for applications. In order to accomplish this task, two approaches have been tested: firstly, the expression of partial cDNAs, and secondly, the expression of synthetic genes in several host organisms, including bacteria, yeast, plants, insect cells, mammalian cells, and transgenic animals. The experienced problems include genetic instability, limitations of the translational and transcriptional machinery, and low solubility of the produced proteins. Here, an overview of attempts to recombinantly produce spidroins will be given, and advantages and disadvantages of the different approaches and host organisms will be discussed. PMID:23415154

Heidebrecht, Aniela; Scheibel, Thomas

2013-01-01

42

Production And Characterization Of Synthetic Spider Silks Based On Nephila Clavipes Major Ampullate Silk Proteins  

NASA Astrophysics Data System (ADS)

The extraordinary mechanical properties of orb-weaving spider silks have served spiders for over 400 million years. However, only in the late 20th century did we start to understand the molecular nature of spider silk that contributes to its incredible properties as biomaterials. Among all seven types of spider silks, major ampullate silk from typical orb-weaving spiders is the toughest of all, it consists of primarily two proteins: MaSp1 and MaSp2. Variable ratios and conserved motifs of these two proteins in all the native spider silks demonstrate the significant role of MaSp1 and MaSp2 in controlling the mechanical properties of the fiber. The amino acid sequences of the orb weaving spider silk proteins have remained almost unchanged for more than 100 million years. Interestingly, MaSp1 and MaSp2 are the only two components in all studied dragline silk fibers from these spiders. The mechanical properties of native dragline silk vary slightly between species, which are believed to relate to the ratio of MaSp1 to MaSp2 in the silk. Both of these facts clearly indicate the importance of these two proteins to the mechanical properties of the fiber. Various types of synthetic spider silk fibers have been produced and studied in an effort to mass-produce man-made fibers with qualities comparable to native spider silk. To investigate the roles of MaSp1 and MaSp2 in silk fiber, synthetic MaSp1 (major abundant protein in Nephila clavipes major ampullate silks) only fibers, MaSp1/MaSp2 protein mixture fibers and chimeric protein fibers with both MaSp1 and MaSp2 sequence features have been produced and tested for mechanical properties. Solid-State Nuclear Magnetic Resonance was used to characterize the structure of silk fibers and reveal the relation between fiber spatial structure and mechanical properties.

An, Bo

43

Solution structure of eggcase silk protein and its implications for silk fiber formation.  

PubMed

Spider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. They are formed from the natural refolding of water-soluble fibroins with alpha-helical and random coil structures in silk glands into insoluble fibers with mainly beta-structures. The structures of the fibroins at atomic resolution and silk formation mechanism remain largely unknown. Here, we report the 3D structures of individual domains of a approximately 366-kDa eggcase silk protein that consists of 20 identical type 1 repetitive domains, one type 2 repetitive domain, and conserved nonrepetitive N- and C-terminal domains. The structures of the individual domains in solution were determined by using NMR techniques. The domain interactions were investigated by NMR and dynamic light-scattering techniques. The formation of micelles and macroscopic fibers from the domains was examined by electron microscopy. We find that either of the terminal domains covalently linked with at least one repetitive domain spontaneously forms micelle-like structures and can be further transformed into fibers at > or = 37 degrees C and a protein concentration of > 0.1 wt%. Our biophysical and biochemical experiments indicate that the less hydrophilic terminal domains initiate the assembly of the proteins and form the outer layer of the micelles whereas the more hydrophilic repetitive domains are embedded inside to ensure the formation of the micelle-like structures that are the essential intermediates in silk formation. Our results establish the roles of individual silk protein domains in fiber formation and provide the basis for designing miniature fibroins for producing artificial silks. PMID:19458259

Lin, Zhi; Huang, Weidong; Zhang, Jingfeng; Fan, Jing-Song; Yang, Daiwen

2009-05-20

44

Engineered spider silk protein-based composites for drug delivery.  

PubMed

Silk protein-based materials are promising materials for the delivery of drugs and other active ingredients, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) in combination with either a polyester (polycaprolactone) or a polyurethane (pellethane), and their physical properties are described. The release profiles are affected by both the film composition and the presence of enzymes, and release can be observed over a period of several weeks. Such silk-based composites have potential as drug eluting biocompatible coatings or implantable devices. PMID:23881554

Hardy, John G; Leal-Egaña, Aldo; Scheibel, Thomas R

2013-07-23

45

Bioengineered chimeric spider silk-uranium binding proteins.  

PubMed

Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium. PMID:23212989

Krishnaji, Sreevidhya Tarakkad; Kaplan, David L

2012-12-04

46

Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins  

PubMed Central

Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins.

Scheibel, Thomas

2004-01-01

47

Protein Adsorption and Biocompatibility of Porous Silk Fiboin Films  

Microsoft Academic Search

Silk fibroins are widely used in skin wound healing and tissue engineering scaffolds. This study sought to observe proteins adsorption and biocompatibility. Bradford protein assay revealed that the amount of adsorbed proteins was significantly less than that on PVA. The Sodium Dodecyl Sulfate Polyacrylamidege Electrophoresis (SDS-PAGE) indicated that more proteins adsorbed onto PVA than that PSFFs. Identification of adsorbed proteins

ZiLing Sun; Lun Bai; Guoping Guan; KuiHua Zhan; HongQin Dai

2009-01-01

48

Solution behavior of synthetic silk peptides and modified recombinant silk proteins  

NASA Astrophysics Data System (ADS)

Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline ?-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the ?-helical structure and decreases in the ?-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in ?-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties.

Foo, C. Wong Po; Bini, E.; Huang, J.; Lee, S. Y.; Kaplan, D. L.

2006-02-01

49

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering.  

PubMed

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches. PMID:22240510

Patra, Chinmoy; Talukdar, Sarmistha; Novoyatleva, Tatyana; Velagala, Siva R; Mühlfeld, Christian; Kundu, Banani; Kundu, Subhas C; Engel, Felix B

2012-01-10

50

Biomaterials Derived from Silk-Tropoelastin Protein Systems  

PubMed Central

A structural protein blend system based on silkworm silk fibroin and recombinant human tropoelastin is described. Silk fibroin, a semicrystalline fibrous protein with beta-sheet crystals provides mechanical strength and controllable biodegradation, while tropoelastin, a noncrystallizable elastic protein provides elasticity. Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) indicated that silk becomes miscible with tropoelastin at different blend ratios, without macrophase separation. Fourier transform infrared spectroscopy (FTIR) revealed secondary structural changes of the blend system (beta-sheet content) before and after methanol treatment. Atomic Force Microscopy (AFM) nano-indentation demonstrated that blending silk and tropoelastin at different ratios resulted in modification of mechanical features, with resilience from ~68% to ~97%, and elastic modulus between 2~9Mpa, depending on the ratio of the two polymers. Some of these values are close to those of native aortic elastin or elastin-like polypeptides. Significantly, during blending and drying silk-tropoelastin form micro- and nano-scale porous morphologies which promote human mesenchymal stem cell attachment and proliferation. These blends offer a new protein biomaterial system for cell support and tailored biomaterial properties to match mechanical needs.

Hu, Xiao; Wang, Xiuli; Rnjak, Jelena; Weiss, Anthony S.; Kaplan, David L.

2010-01-01

51

High-strength silk protein scaffolds for bone repair.  

PubMed

Biomaterials for bone tissue regeneration represent a major focus of orthopedic research. However, only a handful of polymeric biomaterials are utilized today because of their failure to address critical issues like compressive strength for load-bearing bone grafts. In this study development of a high compressive strength (~13 MPa hydrated state) polymeric bone composite materials is reported, based on silk protein-protein interfacial bonding. Micron-sized silk fibers (10-600 µm) obtained utilizing alkali hydrolysis were used as reinforcement in a compact fiber composite with tunable compressive strength, surface roughness, and porosity based on the fiber length included. A combination of surface roughness, porosity, and scaffold stiffness favored human bone marrow-derived mesenchymal stem cell differentiation toward bone-like tissue in vitro based on biochemical and gene expression for bone markers. Further, minimal in vivo immunomodulatory responses suggested compatibility of the fabricated silk-fiber-reinforced composite matrices for bone engineering applications. PMID:22552231

Mandal, Biman B; Grinberg, Ariela; Gil, Eun Seok; Panilaitis, Bruce; Kaplan, David L

2012-05-02

52

Bilayered vascular grafts based on silk proteins.  

PubMed

A major block in the development of small diameter vascular grafts is achieving suitable blood vessel regeneration while minimizing the risk of thrombosis, intimal hyperplasia, suture retention, and mechanical failure. Silk-based tubular vessels for tissue engineering have been prepared by molding, dipping, electrospinning, or gel spinning, however, further studies are needed to improve the mechanical and blood compatibility properties. In the present study a bilayered vascular graft based on silk fibroin (SF) was developed. The graft was composed of an inner silk fiber-reinforced SF tube containing heparin and a highly porous SF external layer. Compared with previously fabricated SF tubes the fiber-reinforcement provided a comparable or higher mechanical strength, burst pressure, and suture retention strength, as well as mechanical compliance, to saphenous veins for vascular grafts. Heparin release was sustained for at least 1month, affording blood compatibility to the grafts. The outer layer of the grafts prepared through lyophilization had a highly porous structure in which the macropore walls were composed of nanofibers similar to extracellular matrix, which offered an excellent environment for cell growth. In vitro studies showed good cytocompatibility and hemocompatibility. PMID:23851155

Liu, Shanshan; Dong, Chaofei; Lu, Guozhong; Lu, Qiang; Li, Zhanxiong; Kaplan, David L; Zhu, Hesun

2013-07-09

53

Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus.  

PubMed

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers. PMID:18376847

La Mattina, Coby; Reza, Ryan; Hu, Xiaoyi; Falick, Arnold M; Vasanthavada, Keshav; McNary, Shannon; Yee, Russell; Vierra, Craig A

2008-04-01

54

Protein and amino acid composition of silks from the cob weaver, Latrodectus hesperus (black widow).  

PubMed

The silks from the cob weaving spider, Latrodectus hesperus (black widow), have been examined with the goal of expanding our understanding of the relationship between the protein structure and mechanical performance of these unique biomaterials. The scaffolding, dragline and inner egg case silks each appear to be distinct fibers based on mole percent amino acid composition and polypeptide composition. Further, we find that the amino acid composition of dragline and egg case silk are similar to the analogous silks produced by orb weaving spiders, while scaffolding silk may represent a novel silk. The black widow silks are comprised of multiple high molecular weight polypeptides, however, the egg case and scaffolding silks also contain some smaller polypeptides. PMID:10342753

Casem, M L; Turner, D; Houchin, K

55

Bombyx mori silk protein films microprocessing with a nanosecond ultraviolet laser and a femtosecond laser workstation: theory and experiments  

NASA Astrophysics Data System (ADS)

Laser microprocessing of several biopolymers from renewable resources is studied. Three proteinic materials were either extracted from the extracellular matrix like Silk Fibroin/Sericin and collagen, or coming from a commercial source like gelatin. All can find future applications in biomedical experimentation, in particular for cell scaffolding. Films of ˜hundred of microns thick were made by aqueous solution drying and laser irradiation. Attention is paid to the properties making them processable with two laser sources: the ultraviolet and nanosecond (ns) KrF (248 nm) excimer and the infrared and femtosecond (fs) Yb:KGW laser. The UV radiation is absorbed in a one-photon resonant process to yield ablation and the surface foaming characteristics of a laser-induced pressure wave. To the contrary, resonant absorption of the IR photons of the fs laser is not possible and does not take place. However, the high field of the intense I>˜1012 W/cm2 femtosecond laser pulse ionizes the film by the multiphoton absorption followed by the electron impact mechanism, yielding a dense plasma capable to further absorb the incident radiation of the end of the pulse. The theoretical model of this absorption is described in detail, and used to discuss the presented experimental effects (cutting, ablation and foaming) of the fs laser. The ultraviolet laser was used to perform simultaneous multiple spots experiments in which energetic foaming yields melt ejection and filament spinning. Airborne nanosize filaments "horizontally suspended by both ends" (0.25 ?m diameter and 10 ?m length) of silk biopolymer were observed upon irradiation with large fluences.

Lazare, S.; Sionkowska, A.; Zaborowicz, M.; Planecka, A.; Lopez, J.; Dijoux, M.; Louména, C.; Hernandez, M.-C.

2012-01-01

56

Sericin for resistance switching device with multilevel nonvolatile memory.  

PubMed

Resistance switching characteristics of natural sericin protein film is demonstrated for nonvolatile memory application for the first time. Excellent memory characteristics with a resistance OFF/ON ratio larger than 10(6) have been obtained and a multilevel memory based on sericin has been achieved. The environmentally friendly high performance biomaterial based memory devices may hold a place in the future of electronic device development. PMID:23893500

Wang, Hong; Meng, Fanben; Cai, Yurong; Zheng, Liyan; Li, Yuangang; Liu, Yuanjun; Jiang, Yueyue; Wang, Xiaotian; Chen, Xiaodong

2013-07-29

57

Primary structure elements of spider dragline silks and their contribution to protein solubility.  

PubMed

Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins. PMID:15491167

Huemmerich, Daniel; Helsen, Christopher W; Quedzuweit, Susanne; Oschmann, Jan; Rudolph, Rainer; Scheibel, Thomas

2004-10-26

58

Recombinant Dragline Silk-Like Proteins--Expression and Purification  

PubMed Central

Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

Gaines, William A.; Marcotte, William R.

2011-01-01

59

Nanoscale control of silica particle formation via silk-silica fusion proteins for bone regeneration.  

PubMed

The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration. PMID:20976116

Mieszawska, Aneta J; Nadkarni, Lauren D; Perry, Carole C; Kaplan, David L

2010-10-26

60

Nanoscale control of silica particle formation via silk-silica fusion proteins for bone regeneration  

PubMed Central

The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration.

Mieszawska, Aneta J.; Nadkarni, Lauren D.; Perry, Carole C.

2010-01-01

61

Silk proteins stimulate osteoblast differentiation by suppressing the Notch signaling pathway in mesenchymal stem cells.  

PubMed

Silk fibroins are biomaterials that have been applied to surgical sutures, drug delivery systems, food supplements, and tissue engineering. Studies have shown the antiadipogenic effects of silk proteins in 3T3-L1 cells and obese mice. Furthermore, other studies have shown that silk proteins increase osteogenic marker expression in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are often regulated reciprocally, we hypothesized that silk proteins would stimulate osteoblast differentiation. The objective of this study was to evaluate the effects of silk proteins on promoting osteoblast differentiation and identify the underlying mechanism. We showed that silk proteins dose dependently stimulated alkaline phosphatase (ALP) activity, osteoblast differentiation, and induced expression of osteoblast markers in C3H10T1/2 and M2-10B4 multipotent cells. In addition, silk proteins also induced the expression of osteoblast markers in primary rat bone marrow cells isolated from tibiae. Molecular studies showed that silk proteins suppressed the expression of Notch-activated genes and blocked activation of the Notch-specific reporter. Similarly, inhibiting Notch signaling with pharmacologic inhibitors and by small interfering RNA-mediated Notch1 silencing also induced ALP activity and messenger RNA expression. Finally, induction of ALP activity and messenger RNA expression by silk proteins was blunted in Notch1 knock-downed cells, further demonstrating Notch signaling as an important mediator for the pro-osteogenic effects of silk proteins. Taken together, our data suggest that silk proteins may serve as functional foods to promote bone healing and therapeutic interventions for bone fractures and osteoporosis. PMID:23399667

Jung, So-Ra; Song, No-Joon; Yang, Dong Kwon; Cho, Yong-Jun; Kim, Byung-Joon; Hong, Joung-Woo; Yun, Ui Jeong; Jo, Dong-Gyu; Lee, Young Min; Choi, Soo Young; Park, Kye Won

2012-12-20

62

Functionalised Silk Fibres.  

National Technical Information Service (NTIS)

The aim of this project was to understand the structure-properties- performance relationship of honeybee silks and to use this information to guide design of second-generation honeybee silk materials. We demonstrated a single recombinant silk protein coul...

T. Sutherland

2012-01-01

63

Biological responses to spider silk-antibiotic fusion protein  

Microsoft Academic Search

The development of a new generation of multifunctional biomaterials is a\\u000d\\u000a continual goal for the field of materials science. The in vivo\\u000d\\u000a functional behaviour of a new fusion protein that combines the\\u000d\\u000a mechanical properties of spider silk with the antimicrobial properties\\u000d\\u000a of hepcidin was addressed in this study. This new chimeric protein,\\u000d\\u000a termed 6mer + hepcidin, fuses spider dragline consensus

Silvia Gomes; Jabier Gallego-Llamas; Isabel B. Leonor; Joao F. Mano; Rui L. Reis; David L. Kaplan

2012-01-01

64

A Novel Model System for Design of Biomaterials Based on Recombinant Analogs of Spider Silk Proteins  

Microsoft Academic Search

Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate\\u000a glands in spiders and comprises of two major structural proteins—spidroins 1 and 2. The relationship between structure and\\u000a mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly\\u000a using two new recombinant

Vladimir G. Bogush; Olga S. Sokolova; Lyubov I. Davydova; Dmitri V. Klinov; Konstantin V. Sidoruk; Natalya G. Esipova; Tatyana V. Neretina; Igor A. Orchanskyi; Vsevolod Yu Makeev; Vladimir G. Tumanyan; Konstantin V. Shaitan; Vladimir G. Debabov; Mikhail P. Kirpichnikov

2009-01-01

65

Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit.  

PubMed

Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W1, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W1 has a high thermal stability with reversible denaturation at ?71°C and forms self-assembled nanoparticle in near-physiological conditions. W1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation. PMID:23994530

Xu, Lingling; Tremblay, Marie-Laurence; Orrell, Kathleen E; Leclerc, Jérémie; Meng, Qing; Liu, Xiang-Qin; Rainey, Jan K

2013-08-28

66

Biological responses to spider silk-antibiotic fusion protein  

PubMed Central

The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer+hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study mice subcutaneous implants were studied to access the in vivo biological response to the 6mer+hepcidin, which were compared with controls of silk alone (6mer), poly-lactic-glycolic-acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer+hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function.

Gomes, Silvia; Gallego-Llamas, Jabier; Leonor, Isabel B.; Mano, Joao F.; Reis, Rui L.; Kaplan, David L.

2011-01-01

67

Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks.  

PubMed

Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor; however, producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, nuclear magnetic resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both beta-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

Jenkins, Janelle E; Creager, Melinda S; Lewis, Randolph V; Holland, Gregory P; Yarger, Jeffery L

2010-01-11

68

Extraordinary Conservation of Cysteines Among Homologous Chironomus Silk Proteins sp185 and sp220  

Microsoft Academic Search

.   Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every\\u000a 20–28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed

Steven T. Case; Carol Cox; Walter C. Bell; Rosemary T. Hoffman; Jon Martin; Robert Hamilton

1997-01-01

69

Utilizing conformational changes for patterning thin films of recombinant spider silk proteins.  

PubMed

Recombinant spider silk proteins mimicking the properties of dragline silk proteins represent a class of materials that hold great potential for future high-performance applications. Here we explore the self-assembly behavior of a recombinantly produced spider silk protein based on the dragline silk of the Araneus diadematus , eADF4 (C16), by selectively patterning its secondary structure using capillary transfer lithography and solvent-assisted microcontact molding. Two conformational transitions were observed, influenced by initial solvent composition: ?-helix/random coil conformation to a more densely packed ?-sheet conformation (by casting from 1,1,1,3,3,3-hexafluoro-propanol) and moderate initial ?-sheet content to higher ?-sheet content (casting from formic acid). Furthermore, by using the solvent-assisted microcontact molding technique, we were able to achieve a submicrometer spatial resolution and reveal fine details of morphological and mechanical changes in patterned regions and at interfaces. PMID:22947370

Young, Seth L; Gupta, Maneesh; Hanske, Christoph; Fery, Andreas; Scheibel, Thomas; Tsukruk, Vladimir V

2012-09-20

70

Integration of silk protein in organic and light-emitting transistors  

PubMed Central

We present the integration of a natural protein into electronic and optoelectronic devices by using silk fibroin as a thin film dielectric in an organic thin film field-effect transistor (OFET) ad an organic light emitting transistor device (OLET) structures. Both n- (perylene) and p-type (thiophene) silk-based OFETs are demonstrated. The measured electrical characteristics are in agreement with high-efficiency standard organic transistors, namely charge mobility of the order of 10-2 cm2/Vs and on/off ratio of 104. The silk-based optolectronic element is an advanced unipolar n-type OLET that yields a light emission of 100nW.

Capelli, R.; Amsden, J. J.; Generali, G.; Toffanin, S.; Benfenati, V.; Muccini, M.; Kaplan, D. L.; Omenetto, F. G.; Zamboni, R.

2012-01-01

71

The molecular structures of major ampullate silk proteins of the wasp spider, Argiope bruennichi: a second blueprint for synthesizing de novo silk.  

PubMed

The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk. PMID:23262065

Zhang, Yang; Zhao, Ai-Chun; Sima, Yang-Hu; Lu, Cheng; Xiang, Zhong-Huai; Nakagaki, Masao

2012-12-20

72

Optically Transparent Recombinant Silk-Elastinlike Protein Polymer Films  

PubMed Central

Recombinant protein polymers, evaluated extensively as biomaterials for applications in drug delivery and tissue engineering, are rarely reported as being optically transparent. Here we report the notable optical transparency of films composed of a genetically engineered silk-elastinlike protein polymer SELP-47K. SELP-47K films of 100 ?m in thickness display a transmittance of 93% in the wavelength range of 350–800 nm. While covalent cross-linking of SELP-47K via glutaraldehyde decreases its transmittance to 77% at the wavelength of 800 nm, noncovalent cross-linking using methanol slightly increases it to 95%. Non- and covalent cross-linking of SELP-47K films also influences their secondary structures and water contents. Cell viability and proliferation analyses further reveal the excellent cytocompatibility of both non- and covalently cross-linked SELP-47K films. The combination of high optical transparency and cytocompatibility of SELP-47K films, together with their previously reported outstanding mechanical properties, suggests that this protein polymer may be useful in unique, new biomedical applications.

Teng, Weibing; Huang, Yiding; Cappello, Joseph; Wu, Xiaoyi

2011-01-01

73

Recombinant minimalist spider wrapping silk proteins capable of native-like fiber formation.  

PubMed

Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W(1) to W(4). We observed that purified W(2), W(3) and W(4) proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W(4) were ?3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm(-3). The smaller W(2) protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W(1) protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly ?-helical in solution to increasingly ?-sheet in fibers. PMID:23209681

Xu, Lingling; Rainey, Jan K; Meng, Qing; Liu, Xiang-Qin

2012-11-28

74

Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects.  

PubMed

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperature-induced transitions of the various silk structures toward beta-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced beta-sheet formation. PMID:15530023

Dicko, Cedric; Knight, David; Kenney, John M; Vollrath, Fritz

75

Light can transform the secondary structure of silk protein  

NASA Astrophysics Data System (ADS)

Fibroin is the main component of silk and is expected to be used as a novel functional material in medicine and bioelectronics. The main secondary structures of this protein are of the random-coil and the ?-sheet types. In this study, we carried out laser-induced transformation of the secondary structure, from the random-coil type to the ?-sheettype, in solid fibroin films. We prepared two types of fibroin films with the random-coil structure. One is a fibroin film doped with a dye as a photosensitizer with a small amount (1 wt%), and the other is a neat fibroin film. The former was excited at 532 nm and the latter was excited at 266 nm. Irradiations were carried out with fluences much lower than each ablation threshold. The excitation of the dye at 532 nm did not affect the secondary structure of the random-coil type. By contrast, 266-nm laser irradiation, which excites tryptophan (an amino-acid residue) involved in fibroin, created the ?-sheetdomain in the film. The structural transformation was revealed by infrared absorption spectroscopy and atomic force microscopy.

Tsuboi, Y.; Ikejiri, T.; Shiga, S.; Yamada, K.; Itaya, A.

76

Resistance to Toxoplasma gondii infection in mice treated with silk protein by enhanced immune responses.  

PubMed

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4(+) and CD8(+) T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-?, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4(+) and CD8(+) T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection. PMID:22072834

Moon, Joung-Ho; Pyo, Kyoung-Ho; Jung, Bong-Kwang; Chun, Hyang Sook; Chai, Jong-Yil; Shin, Eun-Hee

2011-09-30

77

Resistance to Toxoplasma gondii Infection in Mice Treated with Silk Protein by Enhanced Immune Responses  

PubMed Central

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4+ and CD8+ T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-?, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4+ and CD8+ T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.

Moon, Joung-Ho; Pyo, Kyoung-Ho; Jung, Bong-Kwang; Chun, Hyang Sook; Chai, Jong-Yil

2011-01-01

78

Molecular Evolution of Lepidopteran Silk Proteins: Insights from the Ghost Moth, Hepialus californicus  

PubMed Central

Silk production has independently evolved in numerous arthropod lineages, such as Lepidoptera, the moths and butterflies. Lepidopteran larvae (caterpillars) synthesize silk proteins in modified salivary glands and spin silk fibers into protective tunnels, escape lines, and pupation cocoons. Molecular sequence data for these proteins are necessary to determine critical features of their function and evolution. To this end, we constructed an expression library from the silk glands of the ghost moth, Hepialus californicus, and characterized light chainfibroin and heavy chain fibroin gene transcripts. The predicted H. californicus silk fibroins share many elements with other lepidopteran and trichopteran fibroins, such as conserved placements of cysteine, aromatic, and polar amino acid residues. Further comparative analyses were performed to determine site-specific signatures of selection and to assess whether fibroin genes are informative as phylogenetic markers. We found that purifying selection has constrained mutation within the fibroins and that light chain fibroin is a promising molecular marker. Thus, by characterizing the H. californicus fibroins, we identified key functional amino acids and gained insight into the evolutionary processes that have shaped these adaptive molecules.

Mita, Kazuei; Sehnal, Frantisek; Hayashi, Cheryl Y.

2010-01-01

79

Tunable Self-Assembly of Genetically Engineered Silk-Elastin-Like Protein Polymers  

PubMed Central

Silk-elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Due to the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk to elastin blocks in a SELP’s repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.

Xia, Xiao-Xia; Xu, Qiaobing; Hu, Xiao; Qin, Guokui; Kaplan, David L.

2011-01-01

80

Tunable self-assembly of genetically engineered silk--elastin-like protein polymers.  

PubMed

Silk--elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Because of the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk-to-elastin blocks in a SELP's repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels, and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering, and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering. PMID:21955178

Xia, Xiao-Xia; Xu, Qiaobing; Hu, Xiao; Qin, Guokui; Kaplan, David L

2011-09-30

81

Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber  

PubMed Central

Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications.

Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L.; Lee, Sang Yup

2010-01-01

82

Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.  

PubMed

Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L; Lee, Sang Yup

2010-07-26

83

Synthetic spider silk fibers spun from Pyriform Spidroin 2, a glue silk protein discovered in orb-weaving spider attachment discs.  

PubMed

Spider attachment disc silk fibers are spun into a viscous liquid that rapidly solidifies, gluing dragline silk fibers to substrates for locomotion or web construction. Here we report the identification and artificial spinning of a novel attachment disc glue silk fibroin, Pyriform Spidroin 2 (PySp2), from the golden orb weaver Nephila clavipes . MS studies support PySp2 is a constituent of the pyriform gland that is spun into attachment discs. Analysis of the PySp2 protein architecture reveals sequence divergence relative to the other silk family members, including the cob weaver glue silk fibroin PySp1. PySp2 contains internal block repeats that consist of two subrepeat units: one dominated by Ser, Gln, and Ala and the other Pro-rich. Artificial spinning of recombinant PySp2 truncations shows that the Ser-Gln-Ala-rich subrepeat is sufficient for the assembly of polymeric subunits and subsequent fiber formation. These studies support that both orb- and cob-weaving spiders have evolved highly polar block-repeat sequences with the ability to self-assemble into fibers, suggesting a strategy to allow fiber fabrication in the liquid environment of the attachment discs. PMID:21053953

Geurts, Paul; Zhao, Liang; Hsia, Yang; Gnesa, Eric; Tang, Simon; Jeffery, Felicia; La Mattina, Coby; Franz, Andreas; Larkin, Leah; Vierra, Craig

2010-11-05

84

Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori  

SciTech Connect

The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209 bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25 bp fragment. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

Liu Yan [College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027 (China); Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Yu Lian [College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027 (China); Zhejiang Province Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029 (China); Guo Xiuyang [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo Tingqing [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Wang Shengpeng [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Lu Changde [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)]. E-mail: cdlu@sibs.ac.cn

2006-03-31

85

Silk protein based hybrid photonic-plasmonic crystal.  

PubMed

We propose a biocompatible hybrid photonic platform incorporating a 3D silk inverse opal (SIO) crystal and a 2D plasmonic crystal formed on the top surface of the SIO. This hybrid photonic-plasmonic crystal (HPPC) structure simultaneously exhibits both an extraordinary transmission and a pseudo-photonic band-gap in its transmission spectrum. We demonstrate the use of the HPPC as a refractive index (RI) sensor. By performing a multispectral analysis to analyze the RI value, a sensitivity of 200,000 nm·?%T/RIU (refractive index unit) is achieved. PMID:23571980

Kim, Sunghwan; Mitropoulos, Alexander N; Spitzberg, Joshua D; Kaplan, David L; Omenetto, Fiorenzo G

2013-04-01

86

Immobilization of sericin molecules via amorphous carbon plasma modified-polystyrene dish for serum-free culture  

NASA Astrophysics Data System (ADS)

In this study, we focused on sericin hydrolysates, originating from silkworm used in serum-free human bone marrow-derived mesenchymal stem cells (hBM-MSCs) culture. We reported the effect of a covalent linkage between a bioactive protein molecule and polystyrene dish surface via a carbon intermediate layer which can slow down the release rate of protein compounds into the phosphate buffer saline (PBS) solution. Films of amorphous carbon (a-C) and functionalized-carbon were deposited on PS culture dish surfaces by using a DC magnetron sputtering system and RF PECVD system. We found that a-C based-films can increase the hydrophilicity and biocompatibility of polystyrene (PS) dishes, especially a-C films and a-C:N2 films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiOx films), the cells showed a ragged and unattached boundary resulting from the presence of surface silanol groups. For the UV-vis absorbance, all carbon modified-PS dishes showed a lower release rate of sericin molecules into PBS solution than PS control. This revealed that the functionalized carbon could be enhanced by specific binding properties with given molecules. The carbon-coated PS dishes grafting with sericin protein were used in a serum-free condition. We also found that hBM-MSCs have higher percentage of proliferated cells at day 7 for the modified dishes with carbon films and coated with sericin than the PS control coated with sericin. The physical film properties were measured by atomic force microscopy (AFM), scanning electron microscope (SEM) and contact angle measurement. The presence of NH2 groups of sericin compounds on the PS dish was revealed by Fourier transform infrared spectroscopy (FTIR). The stability of covalent bonds of sericin molecules after washing out ungrafted sericin was confirmed by X-ray photoelectron spectroscopy (XPS).

Tunma, Somruthai; Song, Doo-Hoon; Kim, Si-Eun; Kim, Kyoung-Nam; Han, Jeon-Geon; Boonyawan, Dheerawan

2013-10-01

87

Non-bioengineered silk gland fibroin protein: characterization and evaluation of matrices for potential tissue engineering applications.  

PubMed

The possibility of using wild non-mulberry silk protein as a biopolymer remains unexplored compared to domesticated mulberry silk protein. One of the main reasons for this was for not having any suitable method of extraction of silk protein fibroin from cocoons and silk glands. In this study non-bioengineered non-mulberry silk gland fibroin protein from tropical tasar silkworm Antheraea mylitta, is regenerated and characterized using 1% (w/v) sodium dodecyl sulfate (SDS). The new technique is important and unique because it uses a mild surfactant for fibroin dissolution and is advantageous over other previous reported techniques using chaotropic salts. Fabricated fibroin films are smooth as confirmed by atomic force microscopy. Circular dichroism spectrometry along with Fourier transformed infrared spectroscopy and X-ray diffraction reveal random coil/alpha-helix conformations in regenerated fibroin which transform to beta-sheets, resulting in crystalline structure and protein insolubility through ethanol treatment. Differential scanning calorimetry shows an increase in glass transition (Tg) temperature and enhanced degradation temperature on alcohol treatment. Enhanced cell attachment and viability of AH927 feline fibroblasts were observed on fibroin matrices. Higher mechanical strength along with controllable water stability of regenerated gland fibroin films make non-mulberry Indian tropical tasar silk gland fibroin protein a promising biomaterial for tissue engineering applications. PMID:18383269

Mandal, Biman B; Kundu, Subhas C

2008-08-15

88

Air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins.  

PubMed

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly ?-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable ?-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols. PMID:23685883

Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas

2013-05-08

89

A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins.  

PubMed

Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate glands in spiders and comprises of two major structural proteins--spidroins 1 and 2. The relationship between structure and mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly using two new recombinant analogs of spidroins 1 and 2. The artificial genes sequence of the hydrophobic core regions of spidroin 1 and 2 have been designed using computer analysis of existing databases and mathematical modeling. Both proteins were expressed in Pichia pastoris and purified using a cation exchange chromatography. Despite the absence of hydrophilic N- and C-termini, both purified proteins spontaneously formed the nanofibrils and round micelles of about 1 microm in aqueous solutions. The electron microscopy study has revealed the helical structure of a nanofibril with a repeating motif of 40 nm. Using the electrospinning, the thin films with an antiparallel beta-sheet structure were produced. In summary, we were able to obtain artificial structures with characteristics that are perspective for further biomedical applications, such as producing three-dimensional matrices for tissue engineering and drug delivery. PMID:18839314

Bogush, Vladimir G; Sokolova, Olga S; Davydova, Lyubov I; Klinov, Dmitri V; Sidoruk, Konstantin V; Esipova, Natalya G; Neretina, Tatyana V; Orchanskyi, Igor A; Makeev, Vsevolod Yu; Tumanyan, Vladimir G; Shaitan, Konstantin V; Debabov, Vladimir G; Kirpichnikov, Mikhail P

2008-10-07

90

Voltammetric determination of sericin based on its interaction with carmine  

Microsoft Academic Search

A simple yet sensitive method is developed for the determination of sericin using voltammetry based on the interaction between sericin and carmine for the first time. In the absence of sericin, carmine has a pair of well-defined redox peaks in a pH 1.81 Britton–Robinson buffer solution. Although no new redox peaks appear upon the addition of sericin into a carmine

Ming-Ming Ma; Jun-Feng Song

2009-01-01

91

Non-bioengineered silk fibroin protein 3D scaffolds for potential biotechnological and tissue engineering applications.  

PubMed

This paper describes a new source for fabricating high-strength, non-bioengineered silk gland fibroin 3D scaffolds from Indian tropical tasar silkworm, Antheraea mylitta using SDS for dissolution. The scaffolds were fabricated by freeze drying at different prefreezing temperatures for pore size and porosity optimization. Superior mechanical properties with compressive strength in the range of 972 kPa were observed. The matrices were degraded by proteases within 28 d of incubation. Biocompatibility was assessed by feline fibroblast culture in vitro and confocal microscopy further confirmed adherence, spreading, and proliferation of primary dermal fibroblasts. Results indicate nonmulberry 3D silk gland fibroin protein as an inexpensive, high-strength, slow biodegradable, biocompatible, and alternative natural biomaterial. [Figure: see text]. PMID:18702171

Mandal, Biman B; Kundu, Subhas C

2008-09-01

92

Dielectric breakdown strength of regenerated silk fibroin films as a function of protein conformation.  

PubMed

Derived from Bombyx mori cocoons, regenerated silk fibroin (RSF) exhibits excellent biocompatibility, high toughness, and tailorable biodegradability. Additionally, RSF materials are flexible, optically clear, easily patterned with nanoscale features, and may be doped with a variety bioactive species. This unique combination of properties has led to increased interest in the use of RSF in sustainable and biocompatible electronic devices. In order to explore the applicability of this biopolymer to the development of future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a function of protein conformation. The application of processing conditions that increased ?-sheet content (as determined by FTIR analysis) and produced films in the silk II structure resulted in RSF materials with improved Ebd with values reaching up to 400 V/?m. PMID:23987229

Dickerson, Matthew B; Fillery, Scott P; Koerner, Hilmar; Singh, Kristi M; Martinick, Katie; Drummy, Lawrence F; Durstock, Michael F; Vaia, Richard A; Omenetto, Fiorenzo G; Kaplan, David L; Naik, Rajesh R

2013-09-17

93

Differential polymerization of the two main protein components of dragline silk during fibre spinning  

NASA Astrophysics Data System (ADS)

Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3,6,7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk.

Sponner, Alexander; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus

2005-10-01

94

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.  

PubMed

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method. PMID:17525867

Zhang, Yuansong; Hu, Junhua; Miao, Yungen; Zhao, Aichun; Zhao, Tianfu; Wu, Dayang; Liang, Liefeng; Miikura, Ayumi; Shiomi, Kunihiro; Kajiura, Zenta; Nakagaki, Masao

2007-05-25

95

Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders  

Microsoft Academic Search

The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and\\/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera

Catherine L. Craig; Christian Riekel

2002-01-01

96

Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications.  

PubMed

Spider dragline silk is an outstanding material made up of unique proteins-spidroins. Analysis of the amino acid sequences of full-length spidroins reveals a tripartite composition: an N-terminal non-repetitive domain, a highly repetitive central part composed of approximately 100 polyalanine/glycine rich co-segments and a C-terminal non-repetitive domain. Recent molecular data on the terminal domains suggest that these have different functions. The composite nature of spidroins allows for recombinant production of individual and combined regions. Miniaturized spidroins designed by linking the terminal domains with a limited number of repetitive segments recapitulate the properties of native spidroins to a surprisingly large extent, provided that they are produced and isolated in a manner that retains water solubility until fibre formation is triggered. Biocompatibility studies in cell culture or in vivo of native and recombinant spider silk indicate that they are surprisingly well tolerated, suggesting that recombinant spider silk has potential for biomedical applications. PMID:20668909

Rising, Anna; Widhe, Mona; Johansson, Jan; Hedhammar, My

2010-07-29

97

Engineered silk fibroin protein 3D matrices for in vitro tumor model.  

PubMed

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs. PMID:21167597

Talukdar, Sarmistha; Mandal, Mahitosh; Hutmacher, Dietmar W; Russell, Pamela J; Soekmadji, Carolina; Kundu, Subhas C

2010-12-16

98

A novel marine silk.  

PubMed

The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex ?-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements. PMID:22057952

Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz

2011-11-05

99

A novel marine silk  

NASA Astrophysics Data System (ADS)

The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex ?-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements.

Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz

2012-01-01

100

Histochemical and ultrastructural evidence of lipid secretion by the silk gland of the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae).  

PubMed

The silk gland in Lepidoptera larvae is responsible for the silk production used for shelter or cocoon construction. The secretion of fibroin and sericin by the different silk gland regions are well established. There are few attempts to detect lipid components in the insect silk secretion, although the presence of such element may contribute to the resistance of the shelter to wet environment. This study characterizes the glandular region and detects the presence of lipid components in the secretion of the silk gland of Diatraea saccharalis(Fabricius). The silk gland was submitted to histochemical procedure for lipid detection or conventionally prepared for ultrastructural analyses. Lipid droplets were histochemically detected in both the apical cytoplasm of cell of the anterior region and in the lumen among the microvilli. Ultrastructural analyses of the anterior region showed lipid material, visualized as myelin-like structures within the vesicular Golgi complex and in the apical secretory globules, mixed up with the sericin; similar material was observed into the lumen, adjacent to the microvilli. Lipids were not detected in the cells neither in the lumen of the posterior region. Our results suggest that the silk produced by D. saccharalis has a minor lipid content that is secreted by the anterior region together with the sericin. PMID:18060296

Victoriano, Eliane; Pinheiro, Daniela O; Gregório, Elisa A

101

Physical Crosslinking Modulates Sustained Drug Release from Recombinant Silk-Elastinlike Protein Polymer for Ophthalmic Applications  

PubMed Central

We evaluated the drug release capability of optically transparent recombinant silk-elastinlike protein polymer, SELP-47K, films to sustainably deliver the common ocular antibiotic, ciprofloxacin. The ciprofloxacin release kinetics from drug-loaded SELP-47K films treated with ethanol or methanol vapor to induce different densities of physical crosslinking was investigated. Additionally, the drug-loaded protein films were embedded in a protein polymer coating to further prolong the release of the drug. Drug-loaded SELP-47K films released ciprofloxacin for up to 132 hours with near first-order release kinetics. Polymer coating of drug-loaded films prolonged drug release for up to 220 hours. The antimicrobial activity of ciprofloxacin released from the drug delivery matrices was not impaired by the film casting process or the ethanol or methanol treatments. The mechanism of drug release was elucidated by analyzing the physical properties of the film specimens, including equilibrium swelling, soluble fraction, surface roughness and hydrophobicity. Additionally, the conformation of the SELP-47K and its physical crosslinks in the films was analyzed by FTIR and Raman spectroscopy. A three-parameter physics based model accurately described the release rates observed for the various film and coating treatments and attributed the effects to the degree of physical crosslinking of the films and to an increasing affinity of the drug with the polymer network. Together, these results indicate that optically transparent silk-elastinlike protein films may be attractive material candidates for novel ophthalmic drug delivery devices.

Teng, Weibing; Cappello, Joseph; Wu, Xiaoyi

2011-01-01

102

A Complete Recombinant Silk-Elastinlike Protein-Based Tissue Scaffold  

PubMed Central

Due to their improved biocompatibility and specificity over synthetic materials, protein-based biomaterials, either derived from natural sources or genetically engineered, have been widely fabricated into nanofibrous scaffolds for tissue engineering applications. However, their inferior mechanical properties often require the reinforcement of protein-based tissue scaffolds using synthetic polymers. In this study, we report the electrospinning of a completely recombinant silk-elastinlike protein-based tissue scaffold with excellent mechanical properties and biocompatibility. In particular, SELP-47K containing tandemly repeated polypeptide sequences derived from native silk and elastin was electrospun into nanofibrous scaffolds, and stabilized via chemical vapor treatment and mechanical preconditioning. When fully hydrated in 1x PBS at 37 °C, mechanically preconditioned SELP-47K scaffolds displayed elastic moduli of 3.4 to 13.2 MPa, ultimate tensile strengths of 5.7 to 13.5 MPa, deformabilities of 100 to 130% strain, and resilience of 80.6 to 86.9%, closely matching or exceeding those of protein-synthetic blend polymeric scaffolds. Additionally, SELP-47K nanofibrous scaffolds promoted cell attachment and growth demonstrating their in vitro biocompatibility.

Qiu, Weiguo; Huang, Yiding; Teng, Weibing; Cohn, Celine M.; Cappello, Joseph; Wu, Xiaoyi

2010-01-01

103

Effects of degumming conditions on electro-spinning rate of regenerated silk.  

PubMed

Electro-spun silk webs are potentially good candidates as tissue engineering scaffolds owing to their good bio- and cyto-compatibility. However, the low fabrication rate of electro-spun silk mats has been one of the obstacles in the mass production of such nanofibrous silk mats in applications to the biomedical field. In this study, the effects of degumming ratio and silk concentration on the electro-spinning process were investigated by using regenerated silk with different residual sericin contents and different silk concentrations in terms of the morphology and structure of the electro-spun silk web. The rate of production of electro-spun silk mats could be increased by approximately 5 fold at a degumming ratio of 19.5%. The electro-spinning rate of silk was affected by two main factors: (1) dope solution viscosity and (2) degumming ratio of silk. The conductivity of the silk dope solution, however, had little effects on the electro-spinning of regenerated silk. A constant spun fiber morphology was observed within the electro-spinning rate range (0.3-1.4ml/h). Fourier transform infrared spectroscopy showed that partial ?-sheet crystallization occurred during electro-spinning. The molecular conformation was relatively unaffected by the electro-spinning rate of silk. PMID:23817099

Yoon, Kyunghwan; Lee, Ha Ni; Ki, Chang Seok; Fang, Dufei; Hsiao, Benjamin S; Chu, Benjamin; Um, In Chul

2013-06-28

104

Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.  

PubMed

Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSps) that consist of a large repeat array flanked by nonrepetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species. PMID:18828837

Gaines, W A; Marcotte, W R

2008-09-01

105

Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells  

Microsoft Academic Search

Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk

Anthoula Lazaris; Steven Arcidiacono; Yue Huang; Jiang-Feng Zhou; François Duguay; Nathalie Chretien; Elizabeth A. Welsh; Jason W. Soares; Costas N. Karatzas

2002-01-01

106

Spidroin N-terminal Domain Promotes a pH-dependent Association of Silk Proteins during Self-assembly*  

PubMed Central

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.

Gaines, William A.; Sehorn, Michael G.; Marcotte, William R.

2010-01-01

107

Ancient properties of spider silks revealed by the complete gene sequence of the prey-wrapping silk protein (AcSp1).  

PubMed

Spider silk fibers have impressive mechanical properties and are primarily composed of highly repetitive structural proteins (termed spidroins) encoded by a single gene family. Most characterized spidroin genes are incompletely known because of their extreme size (typically >9 kb) and repetitiveness, limiting understanding of the evolutionary processes that gave rise to their unusual gene architectures. The only complete spidroin genes characterized thus far form the dragline in the Western black widow, Latrodectus hesperus. Here, we describe the first complete gene sequence encoding the aciniform spidroin AcSp1, the primary component of spider prey-wrapping fibers. L. hesperus AcSp1 contains a single enormous (?19 kb) exon. The AcSp1 repeat sequence is exceptionally conserved between two widow species (?94% identity) and between widows and distantly related orb-weavers (?30% identity), consistent with a history of strong purifying selection on its amino acid sequence. Furthermore, the 16 repeats (each 371-375 amino acids long) found in black widow AcSp1 are, on average, >99% identical at the nucleotide level. A combination of stabilizing selection on amino acid sequence, selection on silent sites, and intragenic recombination likely explains the extreme homogenization of AcSp1 repeats. In addition, phylogenetic analyses of spidroin paralogs support a gene duplication event occurring concomitantly with specialization of the aciniform glands and the tubuliform glands, which synthesize egg-case silk. With repeats that are dramatically different in length and amino acid composition from dragline spidroins, our L. hesperus AcSp1 expands the knowledge base for developing silk-based biomimetic technologies. PMID:23155003

Ayoub, Nadia A; Garb, Jessica E; Kuelbs, Amanda; Hayashi, Cheryl Y

2012-11-15

108

Ancient Properties of Spider Silks Revealed by the Complete Gene Sequence of the Prey-Wrapping Silk Protein (AcSp1)  

PubMed Central

Spider silk fibers have impressive mechanical properties and are primarily composed of highly repetitive structural proteins (termed spidroins) encoded by a single gene family. Most characterized spidroin genes are incompletely known because of their extreme size (typically >9 kb) and repetitiveness, limiting understanding of the evolutionary processes that gave rise to their unusual gene architectures. The only complete spidroin genes characterized thus far form the dragline in the Western black widow, Latrodectus hesperus. Here, we describe the first complete gene sequence encoding the aciniform spidroin AcSp1, the primary component of spider prey-wrapping fibers. L. hesperus AcSp1 contains a single enormous (?19 kb) exon. The AcSp1 repeat sequence is exceptionally conserved between two widow species (?94% identity) and between widows and distantly related orb-weavers (?30% identity), consistent with a history of strong purifying selection on its amino acid sequence. Furthermore, the 16 repeats (each 371–375 amino acids long) found in black widow AcSp1 are, on average, >99% identical at the nucleotide level. A combination of stabilizing selection on amino acid sequence, selection on silent sites, and intragenic recombination likely explains the extreme homogenization of AcSp1 repeats. In addition, phylogenetic analyses of spidroin paralogs support a gene duplication event occurring concomitantly with specialization of the aciniform glands and the tubuliform glands, which synthesize egg-case silk. With repeats that are dramatically different in length and amino acid composition from dragline spidroins, our L. hesperus AcSp1 expands the knowledge base for developing silk-based biomimetic technologies.

Ayoub, Nadia A.; Garb, Jessica E.; Kuelbs, Amanda; Hayashi, Cheryl Y.

2013-01-01

109

The elaborate structure of spider silk  

PubMed Central

Biomaterials, having evolved over millions of years, often exceed man-made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable. This article focuses on the structure-function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products.

Romer, Lin

2008-01-01

110

Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons.  

PubMed

The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-?, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-?, IL-1? and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair. PMID:24058626

Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

2013-09-13

111

Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons  

PubMed Central

The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from “Sericin Hope” silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-?, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-?, IL-1? and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C.

2013-01-01

112

Regenerated silk fibers: Structural studies and solid state NMR techniques for efficient multiple distance determinations in proteins  

NASA Astrophysics Data System (ADS)

Material Science is the science of understanding the relationship between the molecular level structure of a material and its macroscopic properties. Such research requires both the ability to determine molecular structure and the ability to control and modify the molecular structure. The present research into silks, especially the dragline silk from the spider Nephila clavipes , is occurring at a time when these two criteria are beginning to be met for proteins like spider silk. Genetic engineering has evolved to the point where material scientists have full control over the primary sequence of amino acids that comprise proteins. In addition, solid state nuclear magnetic resonance (NMR) techniques exist which allow us to probe molecular structure. This work applies solid state NMR to the study of the structure of silk fibers. In particular, we focus on techniques of fiber regeneration from solution. The purpose is not only to develop the techniques by which genetically engineered fibers could be spun into fibers for mass production but also as a tool into fundamental silk research. Results on these regenerated fibers show a correlation between the fraction of the silk's alanine residues which are in the ?-sheet conformation and the ultimate tensile strength of the fibers. In addition, in a clever mating of the fiber regeneration technique and the solid state NMR distance measurement experiment, rotational echo double resonance (REDOR), we investigate the supramolecular topology of the alanine ?-sheet crystals. Even though the REDOR technique has failings for the complicated ISn spin systems found in the silk samples, a qualitative analysis does indicate that the ?-sheet crystals are intermolecular. Finally, we investigate a new class of REDOR-like experiments which are designed to overcome the failings of REDOR in ISn spin systems. Experimental data is shown to validate these ideas. An alternate pulse sequence is also introduced and verified with experimental data. This pulse sequence highlights the similarities between multiple quantum NMR and REDOR. From this connection, we name this new class of experiments Multiple Quantum-REDOR. These experiments should allow for efficient simultaneous multiple distance determinations in proteins.

Liivak, Oskar

2000-09-01

113

Science Nation: Got Silk?  

NSDL National Science Digital Library

Spider silk fibers are stronger than almost any other man-made fiber and they're also elastic. There are a lot of potential applications for the fiber, but how do you come up with enough raw material? With help from the National Science Foundation (NSF), researchers have figured out a way to put the spider's silk-making genes into goats in a way that they only make the protein in their milk.

114

Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion  

PubMed Central

Background The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein. Results To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in Salmonella (psicA). LacO operators are placed in various locations in the psicA promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) Latrodectus hesperus dragline silk, Bombyx mori cocoon silk, and Nephila clavipes flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity. Conclusions We show that the natural system encoded in SPI-1 only produces high titers of secreted protein for 4-8 hours when the natural psicA promoter is used to drive expression. Secretion efficiency can be high, but declines for charged or large sequences. A quantitative characterization of these constraints will facilitate the effective use and engineering of this system.

2010-01-01

115

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties.  

PubMed

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers. PMID:22215590

Teulé, Florence; Miao, Yun-Gen; Sohn, Bong-Hee; Kim, Young-Soo; Hull, J Joe; Fraser, Malcolm J; Lewis, Randolph V; Jarvis, Donald L

2012-01-03

116

Genome-based identification of spliceosomal proteins in the silk moth Bombyx mori.  

PubMed

Pre-messenger RNA splicing is a highly conserved eukaryotic cellular function that takes place by way of a large, RNA-protein assembly known as the spliceosome. In the mammalian system, nearly 300 proteins associate with uridine-rich small nuclear (sn)RNAs to form this complex. Some of these splicing factors are ubiquitously present in the spliceosome, whereas others are involved only in the processing of specific transcripts. Several proteomics analyses have delineated the proteins of the spliceosome in several species. In this study, we mine multiple sequence data sets of the silk moth Bombyx mori in an attempt to identify the entire set of known spliceosomal proteins. Five data sets were utilized, including the 3X, 6X, and Build 2.0 genomic contigs as well as the expressed sequence tag and protein libraries. While homologs for 88% of vertebrate splicing factors were delineated in the Bombyx mori genome, there appear to be several spliceosomal polypeptides absent in Bombyx mori and seven additional insect species. This apparent increase in spliceosomal complexity in vertebrates may reflect the tissue-specific and developmental stage-specific alternative pre-mRNA splicing requirements in vertebrates. Phylogenetic analyses of 15 eukaryotic taxa using the core splicing factors suggest that the essential functional units of the pre-mRNA processing machinery have remained highly conserved from yeast to humans. The Sm and LSm proteins are the most conserved, whereas proteins of the U1 small nuclear ribonucleoprotein particle are the most divergent. These data highlight both the differential conservation and relative phylogenetic signals of the essential spliceosomal components throughout evolution. PMID:21104883

Somarelli, Jason A; Mesa, Annia; Fuller, Myron E; Torres, Jacqueline O; Rodriguez, Carol E; Ferrer, Christina M; Herrera, Rene J

2010-12-01

117

Voltammetric determination of sericin based on its interaction with carmine.  

PubMed

A simple yet sensitive method is developed for the determination of sericin using voltammetry based on the interaction between sericin and carmine for the first time. In the absence of sericin, carmine has a pair of well-defined redox peaks in a pH 1.81 Britton-Robinson buffer solution. Although no new redox peaks appear upon the addition of sericin into a carmine solution, the peak currents of the old peaks reduce while the peak potentials shift positively. This observation is attributed to the decrease in the diffusion coefficient and electrode reaction rate constant of carmine in the presence of sericin. A binding mechanism is proposed and discussed, and the binding constant and binding ratio are calculated as 2.32 x 10(6) L mol(-1) and 1:1, respectively. Furthermore, the decrease in the peak currents is found proportional to the sericin concentration in the range of 32.0-800.0 microg mL(-1) with a detection limit of 13.52 microg mL(-1). The method is further applied to the determination of sericin in degumming wastewater with satisfied average recoveries from 96.7 to 103.3%. The results are in good agreement with those obtained by the conventional Coomassie brilliant blue G-250 spectrophotometric method. PMID:19782207

Ma, Ming-Ming; Song, Jun-Feng

2009-07-01

118

Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene  

PubMed Central

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering.

Sakudoh, Takashi; Sezutsu, Hideki; Nakashima, Takeharu; Kobayashi, Isao; Fujimoto, Hirofumi; Uchino, Keiro; Banno, Yutaka; Iwano, Hidetoshi; Maekawa, Hideaki; Tamura, Toshiki; Kataoka, Hiroshi; Tsuchida, Kozo

2007-01-01

119

Non-mulberry silk gland fibroin protein 3-D scaffold for enhanced differentiation of human mesenchymal stem cells into osteocytes.  

PubMed

This study investigates the potential of three-dimensional (3-D) scaffolds of wild non-mulberry tropical tasar silk gland fibroin protein as substratum for osteogenic differentiation of human mesenchymal stem cells (hMSCs). The novelty of the study lies in the fabrication of scaffolds from non-bioengineered silk fibroin directly extracted from the glands of non-mulberry tropical tasar silkworms using sodium dodecyl sulfate dissolution protocol and its osteogenic application using single- and double-seeding methods. The scaffolds were mechanically robust and showed homogenous pore distribution within the scaffold. hMSCs were seeded on the scaffolds and were cultured for up to 28days under static conditions in osteogenic media. Osteogenic differentiation of hMSCs seeded on fibroin scaffolds resulted in extensive mineralization with the formation of large calcium nodules, higher alkaline phosphatase activity and intense von Kossa staining. Real-time studies revealed higher transcript levels for osteopontin (OS) and bone sialoprotein (IBSP) under double-seeded conditions as compared to single-seeded scaffolds. Histological analysis showed the development of osteoblastic cells and large calcified nodules. The development and spreading of nuclei and actin filaments on fibroin matrices were revealed through confocal studies. The results suggest the suitability of non-mulberry silk-fibroin protein 3-D scaffolds as natural biomaterial for potential in vitro bone-tissue engineering applications. PMID:19345621

Mandal, Biman B; Kundu, S C

2009-03-05

120

Mechanical Improvements to Reinforced Porous Silk Scaffolds  

PubMed Central

Load bearing porous biodegradable scaffolds are required to engineer functional tissues such as bone. Mechanical improvements to porogen leached scaffolds prepared from silk proteins were systematically studied through the addition of silk particles in combination with silk solution concentration, exploiting interfacial compatibility between the two components. Solvent solutions of silk up to 32 w/v% were successfully prepared in hexafluoroisopropanaol (HFIP) for the study. The mechanical properties of the reinforced silk scaffolds correlated to the material density and matched by a power law relationship, independent of the ratio of silk particles to matrix. These results were similar to the relationships previously shown for cancellous bone. The mechanism behind the increased mechanical properties was a densification effect, and not the effect of including stiffer silk particles into the softer silk continuous matrix. A continuous interface between the silk matrix and the silk particles, as well as homogeneous distribution of the silk particles within the matrix were observed. Furthermore, we note that the roughness of the pore walls was controllable by varying the ratio of particles matrix, providing a route to control topography. The rate of proteolytic hydrolysis of the scaffolds decreased with increase in mass of silk used in the matrix and with increasing silk particle content.

Gil, Eun Seok; Kluge, Jonathan A.; Rockwood, Danielle N.; Rajkhowa, Rangam; Wang, Lijing; Wang, Xungai; Kaplan, David L

2012-01-01

121

Structural Origins of Silk Piezoelectricity  

PubMed Central

Uniaxially oriented, piezoelectric silk films were prepared by a two-step method that involved: (1) air drying aqueous, regenerated silk fibroin solutions into films, and (2) drawing the silk films to a desired draw ratio. The utility of two different drawing techniques, zone drawing and water immersion drawing were investigated for processing the silk for piezoelectric studies. Silk films zone drawn to a ratio of ?= 2.7 displayed relatively high dynamic shear piezoelectric coefficients of d14 = ?1.5 pC/N, corresponding to over two orders of magnitude increase in d14 due to film drawing. A strong correlation was observed between the increase in the silk II, ?-sheet content with increasing draw ratio measured by FTIR spectroscopy (C?? e2.5 ?), the concomitant increasing degree of orientation of ?-sheet crystals detected via WAXD (FWHM = 0.22° for ?= 2.7), and the improvement in silk piezoelectricity (d14? e2.4 ?). Water immersion drawing led to a predominantly silk I structure with a low degree of orientation (FWHM = 75°) and a much weaker piezoelectric response compared to zone drawing. Similarly, increasing the ?-sheet crystallinity without inducing crystal alignment, e.g. by methanol treatment, did not result in a significant enhancement of silk piezoelectricity. Overall, a combination of a high degree of silk II, ?-sheet crystallinity and crystalline orientation are prerequisites for a strong piezoelectric effect in silk. Further understanding of the structural origins of silk piezoelectricity will provide important options for future biotechnological and biomedical applications of this protein.

Yucel, Tuna; Cebe, Peggy

2012-01-01

122

Structural Origins of Silk Piezoelectricity.  

PubMed

Uniaxially oriented, piezoelectric silk films were prepared by a two-step method that involved: (1) air drying aqueous, regenerated silk fibroin solutions into films, and (2) drawing the silk films to a desired draw ratio. The utility of two different drawing techniques, zone drawing and water immersion drawing were investigated for processing the silk for piezoelectric studies. Silk films zone drawn to a ratio of ?= 2.7 displayed relatively high dynamic shear piezoelectric coefficients of d(14) = -1.5 pC/N, corresponding to over two orders of magnitude increase in d(14) due to film drawing. A strong correlation was observed between the increase in the silk II, ?-sheet content with increasing draw ratio measured by FTIR spectroscopy (C(?)? e(2.5) (?)), the concomitant increasing degree of orientation of ?-sheet crystals detected via WAXD (FWHM = 0.22° for ?= 2.7), and the improvement in silk piezoelectricity (d(14)? e(2.4) (?)). Water immersion drawing led to a predominantly silk I structure with a low degree of orientation (FWHM = 75°) and a much weaker piezoelectric response compared to zone drawing. Similarly, increasing the ?-sheet crystallinity without inducing crystal alignment, e.g. by methanol treatment, did not result in a significant enhancement of silk piezoelectricity. Overall, a combination of a high degree of silk II, ?-sheet crystallinity and crystalline orientation are prerequisites for a strong piezoelectric effect in silk. Further understanding of the structural origins of silk piezoelectricity will provide important options for future biotechnological and biomedical applications of this protein. PMID:23335872

Yucel, Tuna; Cebe, Peggy; Kaplan, David L

2011-01-13

123

A structural view on spider silk proteins and their role in fiber assembly.  

PubMed

Spider silk is the toughest known biomaterial and even outrivals modern synthetic high-performance materials. The question of understanding fiber formation is how the spider can prevent premature and fatal aggregation processes inside its own body and how the chemical and mechanical stimuli used to induce the fiber formation process translate into structural changes of the silk material, finally leading to controlled and irreversible aggregation. Here, the focus will be on the structure and function of the highly conserved N-domains and C-terminal domains of spider dragline silk which, unlike the very long repetitive sequence elements, adopt a folded conformation in solution and are therefore able to control intermolecular interactions and aggregation between other spider silk molecules. The structures of these domains add valuable details for the construction of a molecular picture of the complicated and highly optimized silk assembly process that might be beneficial for large-scale in vitro fiber formation attempts with recombinant silk material. PMID:22570231

Hagn, Franz

2012-05-08

124

Characterization of iron oxide nanoparticles in structural silk-elastinlike protein polymer  

NASA Astrophysics Data System (ADS)

The structure of silk elastin-like protein (SELP) block copolymers containing Fe3O4 magnetic nanoparticles are investigated. These materials have potential applications for hyperthermia cancer therapy. SELPs undergo a gel transition at physiological temperatures, which can be used to localize delivery of nanoparticles at tumor sites. Vibrating sample magnetometry (VSM), transmission electron microscopy (TEM), and small angle neutron scattering (SANS) are used to characterize the nanoparticles and the SELP-nanoparticle nanocomposite system. A series of nanoparticles with three different nominal diameters, 30, 50 and 80 nm, were added to 4 and 8 wt.% SELP samples. Different functionalities on the nanoparticle surface affect their interactions with SELP. The 50 nm nanoparticles in SELP exhibit chaining (linear association of the nanoparticles), while the 30 nm nanoparticles are too small and settle out of the polymer mesh and the 80 nm nanoparticles tend to cluster without any regard for SELP structure. The SELP concentration does not have a major affect on nanoparticle behavior in the nanocomposites.

Shih, Jennifer

125

Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR and X-ray Diffraction.  

PubMed

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked ?-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in ?-sheet (poly(Alan) and poly(Gly-Ala)), 31-helix (poly(Gly-Gly-Xaa), and ?-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in ?-sheet (poly(Gly-Ala)) and 31-helical (poly(Gly-Gly-Xaa)) regions, while serine is present in ?-sheet (poly(Gly-Ala-Ser)), 31-helix (poly(Gly-Gly-Ser)), and ?-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

Jenkins, Janelle E; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W; Holland, Gregory P; Yarger, Jeffery L

2013-09-26

126

Functional design of spider's silk  

NASA Astrophysics Data System (ADS)

The orb-web weaving spiders produce a broad range of high performance structural fibers (i.e. silks) with mechanical properties that are superbly matched to their function. Our interest in these materials stems both from an interest in the biology of the spiders and the design of their webs and also from a desire to discover principles of mechanical design of protein-based structural materials that can guide the development of novel bio-engineered materials. All spiders produce silks, but the orb-web weaving spiders are unique in their ability to produce seven different silks, each from distinct gland/spinneret complexes. Considering the wide diversity of spider species, there is likely to be an enormous range of material properties available in spider silk. However, at present, we only have information on two species of spiders, and only two of their seven silks have been studied in any detail. These are: (1) the silk produced by the major ampullate gland, which forms the safety-line or dragline of the spider and also is used to form the frame of its orb-web, and (2) the viscid silk produced by the flagelliform gland, which forms the glue-covered catching spiral of the web. In this paper we describe several aspects of the mechanical design of the dragline and viscid silks produced by the spider Araneus diadematus.

Gosline, John; Guerette, Paul; Ortlepp, Christine

1996-02-01

127

Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion.  

PubMed

Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential, fibroin proteins from Antheraea mylitta and Bombyx mori are immunologically inert and invoke minimal immune response. Stimulation of murine peritoneal macrophages and RAW 264.7 murine macrophages by these fibroin proteins both in solution and in the form of films assayed in terms of nitric oxide and TNFalpha production showed comparable stimulation as in collagen. Kinetics of adhesion of L929 murine fibroblasts, for biocompatibility evaluation, monitored every 4 h from seeding and studied over a period of 24 h, reveal A. mylitta fibroin film to be a better substrate in terms of rapid and easier cellularization. Cell viability studies by MTT assay and flow cytometric analyses indicate the ability of fibroin matrices to support cell growth and proliferation comparable to collagen for long-term culture. This matrix may have potential to serve in those injuries where rapid cellularization is essential. PMID:18322779

Acharya, Chitrangada; Ghosh, Sudip K; Kundu, S C

2008-03-06

128

Silk Fibroin Electrogelation Mechanisms  

PubMed Central

A silk fibroin gel system (e-gel), formed with weak electric fields has potential utility in medical materials and devices. The mechanism of silk e-gel formation was studied to gain additional insight into the process and control of the material properties. Silk fibroin nanoparticles with sizes of several ten nanometers, composed of metastable conformations, were involved in e-gel formation. Under electric fields, the nanoparticles rapidly assembled into larger nano- or microspheres with size ranges from tens nanometers to several microns. Repulsive forces from the negative surface charge of the acidic groups on the protein were screened by the local decrease in solution pH in the vicinity of the positive electrode. By controlling the formation and content of silk fibroin nanoparticles e-gel could be formed even from low concentration silk fibroin solutions (1%). When e-gel was reversed to the solution state, the aggregated nano- and microspheres dispersed into solution, a significant observation related to future applications for this process, such as for drug delivery.

Lu, Qiang; Huang, Yongli; Li, Mingzhong; Zuo, Baoqi; Lu, Shenzhou; Wang, Jiannan; Zhu, Hesun; Kaplan, David L.

2012-01-01

129

Conservation of Essential Design Features in Coiled Coil Silks  

Microsoft Academic Search

Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended b-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small (;30 kDa each) and nonrepetitive and

Tara D. Sutherland; Sarah Weisman; Holly E. Trueman; Alagacone Sriskantha; John W. H. Trueman; Victoria S. Haritos

2007-01-01

130

Comparison of silk-elastinlike protein polymer hydrogel and poloxamer in matrix-mediated gene delivery.  

PubMed

The silk-elastinlike protein polymer, SELP 815K, and poloxomer 407, a commercially available synthetic copolymer, were evaluated to compare their relative performance in matrix-mediated viral gene delivery. Using a xenogenic mouse tumor model of human head and neck squamous cell carcinoma, the efficacy of viral gene-directed enzyme prodrug therapy with these polymers was characterized by viral gene expression in the tumor tissue, tumor size reduction, and survivability with treatment. Viral injection in SELP 815K produced a greater level and more prolonged extent of gene expression in the tumor, a statistically greater tumor size reduction, a longer time until tumor rebound, and a significantly increased survivability, as compared to injection of virus alone or in Poloxamer 407. Safety of treatment with these polymers was evaluated in a non-tumor bearing immunocompetent mouse model. Compared to virus injected alone or in Poloxamer 407, virus injected in SELP 815K had fewer and less severe indications of toxicity related to treatment as assessed by blood analysis, body weight, and histopathology of distant organs and the injection sites. Similar to virus alone or in Poloxamer 407, virus injected in SELP 815K elicited a mild injection site inflammatory response characterized primarily by a mononuclear leukocyte infiltrate and the formation of granulation tissue. Virus injected in SELP 815K resulted in fewer animals with elevated white blood cell counts and a less pronounced local toxicity reaction than was observed with virus in Poloxamer 407. In contrast to virus injected alone or in Poloxamer 407, which were not retained in the injection site tissues beyond week 1, SELP 815K was retained at the injection sites and by the end of the study (week 12), displayed limited absorption, and mild encapsulation. These results demonstrate the benefits of SELP 815K for matrix-mediated gene delivery over the injection of free virus and the injection of virus in Poloxamer 407. Virus in SELP 815K had greater efficacy of tumor suppression, promoted greater levels and greater duration of viral gene expression, and displayed reduced levels of injection site toxicity. Combining these performance and safety benefits with the degree of control with which they can be designed, synthesized and formulated, SELPs continue to show promise for their application in viral gene delivery. PMID:21982738

Price, Robert; Gustafson, Joshua; Greish, Khaled; Cappello, Joseph; McGill, Lawrence; Ghandehari, Hamidreza

2011-10-01

131

Comparison of Silk-Elastinlike Protein Polymer Hydrogel and Poloxamer in Matrix-Mediated Gene Delivery  

PubMed Central

The silk-elastinlike protein polymer, SELP 815K, and poloxomer 407, a commercially available synthetic copolymer, were evaluated to compare their relative performance in matrix mediated viral gene delivery. Using a xenogenic mouse tumor model of human head and neck squamous cell carcinoma, the efficacy of viral gene-directed enzyme prodrug therapy with these polymers was characterized by viral gene expression in the tumor tissue, tumor size reduction, and survivability with treatment. Viral injection in SELP 815K produced a greater level and more prolonged extent of gene expression in the tumor, a statistically greater tumor size reduction, a longer time until tumor rebound, and a significantly increased survivability, as compared to injection of virus alone or in poloxamer 407. Safety of treatment with these polymers was evaluated in a non-tumor bearing immunocompetent mouse model. Compared to virus injected alone or in poloxamer 407, virus injected in SELP 815K had fewer and less severe indications of toxicity related to treatment as assessed by blood analysis, body weight, and histopathology of distant organs and the injection sites. Similar to virus alone or in poloxamer 407, virus injected in SELP 815K elicited a mild injection site inflammatory response characterized primarily by a mononuclear leukocyte infiltrate and the formation of granulation tissue. Virus injected in SELP 815K resulted in fewer animals with elevated white blood cell counts and a less pronounced local toxicity reaction than was observed with virus in poloxamer 407. In contrast to virus injected alone or in poloxamer 407, which were not retained in the injection site tissues beyond week 1, SELP 815K was retained at the injection sites and by the end of the study (week 12), displayed limited absorption, and mild encapsulation. These results demonstrate the benefits of SELP 815K for matrix-mediated gene delivery over the injection of free virus and the injection of virus in Poloxamer 407. Virus in SELP 815K had greater efficacy of tumor suppression, promoted greater levels and greater duration of viral gene expression, and displayed reduced levels of injection site toxicity. Combining these performance and safety benefits with the degree of control with which they can be designed, synthesized and formulated, SELPs continue to show promise for their application in viral gene delivery.

Price, Robert; Gustafson, Joshua; Greish, Khaled; Cappello, Joseph; McGill, Lawrence; Ghandehari, Hamidreza

2011-01-01

132

The molecular structure of spider dragline silk: Folding and orientation of the protein backbone  

PubMed Central

The design principles of spider dragline silk, nature's high-performance fiber, are still largely unknown, in particular for the noncrystalline glycine-rich domains, which form the bulk of the material. Here we apply two-dimensional solid-state NMR to determine the distribution of the backbone torsion angles (?,?) as well as the orientation of the polypeptide backbone toward the fiber at both the glycine and alanine residues. Instead of an “amorphous matrix,” suggested earlier for the glycine-rich domains, these new data indicate that all domains in dragline silk have a preferred secondary structure and are strongly oriented, with the chains predominantly parallel to the fiber. As proposed previously, the alanine residues are predominantly found in a ? sheet conformation. The glycine residues are partly incorporated into the ? sheets and otherwise form helical structures with an approximate 3-fold symmetry.

van Beek, J. D.; Hess, S.; Vollrath, F.; Meier, B. H.

2002-01-01

133

An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing.  

PubMed

In this study, the novel silk fibroin-based bi-layered wound dressing was developed. Wax-coated silk fibroin woven fabric was introduced as a non-adhesive layer while the sponge made of sericin and glutaraldehyde-crosslinked silk fibroin/gelatin was fabricated as a bioactive layer. Wax-coated silk fibroin fabrics showed improved mechanical properties compared with the non-coated fabrics, but less adhesive than the commercial wound dressing mesh. This confirmed by results of peel test on both the partial- and full-thickness wounds. The sericin-silk fibroin/gelatin spongy bioactive layers showed homogeneous porous structure and controllable biodegradation depending on the degree of crosslinking. The bi-layered wound dressings supported the attachment and proliferation of L929 mouse fibroblasts, particularly for the silk fibroin/gelatin ratio of 20/80 and 0.02% GA crosslinked. Furthermore, we proved that the bi-layered wound dressings promoted wound healing in full-thickness wounds, comparing with the clinically used wound dressing. The wounds treated with the bi-layered wound dressings showed the greater extent of wound size reduction, epithelialization, and collagen formation. The superior properties of the silk fibroin-based bi-layered wound dressings compared with those of the clinically used wound dressings were less adhesive and had improved biological functions to promote cell activities and wound healing. This novel bi-layered wound dressing should be a good candidate for the healing of full-thickness wounds. PMID:22771972

Kanokpanont, Sorada; Damrongsakkul, Siriporn; Ratanavaraporn, Juthamas; Aramwit, Pornanong

2012-07-04

134

Invited review nonmulberry silk biopolymers.  

PubMed

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials. PMID:22241173

Kundu, S C; Kundu, Banani; Talukdar, Sarmistha; Bano, Subia; Nayak, Sunita; Kundu, Joydip; Mandal, Biman B; Bhardwaj, Nandana; Botlagunta, Mahendran; Dash, Biraja C; Acharya, Chitrangada; Ghosh, Ananta K

2012-01-12

135

Reproducing natural spider silks' copolymer behavior in synthetic silk mimics.  

PubMed

Dragline silk from orb-weaving spiders is a copolymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia , indicates that MaSp1 proteins are more easily formed into ?-sheet nanostructures, while MaSp2 proteins form random coil and helical structures. To test if this behavior of natural silk proteins could be reproduced by recombinantly produced spider silk mimic protein, recombinant MaSp1/MaSp2 mixed fibers as well as chimeric silk fibers from MaSp1 and MaSp2 sequences in a single protein were produced based on the variable ratio and conserved motifs of MaSp1 and MaSp2 in native silk fiber. Mechanical properties, solid-state NMR, and XRD results of tested synthetic fibers indicate the differing roles of MaSp1 and MaSp2 in the fiber and verify the importance of postspin stretching treatment in helping the fiber to form the proper spatial structure. PMID:23110450

An, Bo; Jenkins, Janelle E; Sampath, Sujatha; Holland, Gregory P; Hinman, Mike; Yarger, Jeffery L; Lewis, Randolph

2012-11-08

136

Silk from Crickets: A New Twist on Spinning  

PubMed Central

Raspy crickets (Orthoptera: Gryllacrididae) are unique among the orthopterans in producing silk, which is used to build shelters. This work studied the material composition and the fabrication of cricket silk for the first time. We examined silk-webs produced in captivity, which comprised cylindrical fibers and flat films. Spectra obtained from micro-Raman experiments indicated that the silk is composed of protein, primarily in a beta-sheet conformation, and that fibers and films are almost identical in terms of amino acid composition and secondary structure. The primary sequences of four silk proteins were identified through a mass spectrometry/cDNA library approach. The most abundant silk protein was large in size (300 and 220 kDa variants), rich in alanine, glycine and serine, and contained repetitive sequence motifs; these are features which are shared with several known beta-sheet forming silk proteins. Convergent evolution at the molecular level contrasts with development by crickets of a novel mechanism for silk fabrication. After secretion of cricket silk proteins by the labial glands they are fabricated into mature silk by the labium-hypopharynx, which is modified to allow the controlled formation of either fibers or films. Protein folding into beta-sheet structure during silk fabrication is not driven by shear forces, as is reported for other silks.

Walker, Andrew A.; Weisman, Sarah; Church, Jeffrey S.; Merritt, David J.; Mudie, Stephen T.; Sutherland, Tara D.

2012-01-01

137

Antheraea pernyi silk fiber: a potential resource for artificially biospinning spider dragline silk.  

PubMed

The outstanding properties of spider dragline silk are likely to be determined by a combination of the primary sequences and the secondary structure of the silk proteins. Antheraea pernyi silk has more similar sequences to spider dragline silk than the silk from its domestic counterpart, Bombyx mori. This makes it much potential as a resource for biospinning spider dragline silk. This paper further verified its possibility as the resource from the mechanical properties and the structures of the A. pernyi silks prepared by forcible reeling. It is surprising that the stress-strain curves of the A. pernyi fibers show similar sigmoidal shape to those of spider dragline silk. Under a controlled reeling speed of 95 mm/s, the breaking energy was 1.04 x 10(5) J/kg, the tensile strength was 639 MPa and the initial modulus was 9.9 GPa. It should be noted that this breaking energy of the A. pernyi silk approaches that of spider dragline silk. The tensile properties, the optical orientation and the beta-sheet structure contents of the silk fibers are remarkably increased by raising the spinning speeds up to 95 mm/s. PMID:20454537

Zhang, Yaopeng; Yang, Hongxia; Shao, Huili; Hu, Xuechao

2010-05-05

138

Antheraea pernyi Silk Fiber: A Potential Resource for Artificially Biospinning Spider Dragline Silk  

PubMed Central

The outstanding properties of spider dragline silk are likely to be determined by a combination of the primary sequences and the secondary structure of the silk proteins. Antheraea pernyi silk has more similar sequences to spider dragline silk than the silk from its domestic counterpart, Bombyx mori. This makes it much potential as a resource for biospinning spider dragline silk. This paper further verified its possibility as the resource from the mechanical properties and the structures of the A. pernyi silks prepared by forcible reeling. It is surprising that the stress-strain curves of the A. pernyi fibers show similar sigmoidal shape to those of spider dragline silk. Under a controlled reeling speed of 95?mm/s, the breaking energy was 1.04 × 105?J/kg, the tensile strength was 639 MPa and the initial modulus was 9.9 GPa. It should be noted that this breaking energy of the A. pernyi silk approaches that of spider dragline silk. The tensile properties, the optical orientation and the ?-sheet structure contents of the silk fibers are remarkably increased by raising the spinning speeds up to 95?mm/s.

Zhang, Yaopeng; Yang, Hongxia; Shao, Huili; Hu, Xuechao

2010-01-01

139

Microdissection of black widow spider silk-producing glands.  

PubMed

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments. PMID:21248709

Jeffery, Felicia; La Mattina, Coby; Tuton-Blasingame, Tiffany; Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-11

140

Microdissection of Black Widow Spider Silk-producing Glands  

PubMed Central

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-01

141

Early Events in the Evolution of Spider Silk Genes  

Microsoft Academic Search

Silk spinning is essential to spider ecology and has had a key role in the expansive diversification of spiders. Silk is composed primarily of proteins called spidroins, which are encoded by a multi-gene family. Spidroins have been studied extensively in the derived clade, Orbiculariae (orb-weavers), from the suborder Araneomorphae (‘true spiders’). Orbicularians produce a suite of different silks, and underlying

James Starrett; Jessica E. Garb; Amanda Kuelbs; Ugochi O. Azubuike; Cheryl Y. Hayashi

2012-01-01

142

Mechanism of silk processing in insects and spiders  

Microsoft Academic Search

Silk spinning by insects and spiders leads to the formation of fibres that exhibit high strength and toughness. The lack of understanding of the protein processing in silk glands has prevented the recapitulation of these properties in vitro from reconstituted or genetically engineered silks. Here we report the identification of emulsion formation and micellar structures from aqueous solutions of reconstituted

Hyoung-Joon Jin; David L. Kaplan

2003-01-01

143

Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes.  

PubMed

Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to beta-structures and alpha-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core. PMID:20214876

Antonenko, Yuri N; Perevoshchikova, Irina V; Davydova, Lyubov I; Agapov, Igor A; Bogush, Vladimir G

2010-03-07

144

Post-secretion processing influences spider silk performance  

PubMed Central

Phenotypic variation facilitates adaptations to novel environments. Silk is an example of a highly variable biomaterial. The two-spidroin (MaSp) model suggests that spider major ampullate (MA) silk is composed of two proteins—MaSp1 predominately contains alanine and glycine and forms strength enhancing ?-sheet crystals, while MaSp2 contains proline and forms elastic spirals. Nonetheless, mechanical properties can vary in spider silks without congruent amino acid compositional changes. We predicted that post-secretion processing causes variation in the mechanical performance of wild MA silk independent of protein composition or spinning speed across 10 species of spider. We used supercontraction to remove post-secretion effects and compared the mechanics of silk in this ‘ground state’ with wild native silks. Native silk mechanics varied less among species compared with ‘ground state’ silks. Variability in the mechanics of ‘ground state’ silks was associated with proline composition. However, variability in native silks did not. We attribute interspecific similarities in the mechanical properties of native silks, regardless of amino acid compositions, to glandular processes altering molecular alignment of the proteins prior to extrusion. Such post-secretion processing may enable MA silk to maintain functionality across environments, facilitating its function as a component of an insect-catching web.

Blamires, Sean J.; Wu, Chung-Lin; Blackledge, Todd A.; Tso, I-Min

2012-01-01

145

Materials fabrication from Bombyx mori silk fibroin  

Microsoft Academic Search

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing

Danielle N Rockwood; Rucsanda C Preda; Tuna Yücel; Xiaoqin Wang; Michael L Lovett; David L Kaplan

2011-01-01

146

Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells  

NASA Astrophysics Data System (ADS)

Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, François; Chretien, Nathalie; Welsh, Elizabeth A.; Soares, Jason W.; Karatzas, Costas N.

2002-01-01

147

Tussah Silk Fibroin Excels Silk Fibroin from the Domesticated Silkworm in Supporting the Development of Neurons  

Microsoft Academic Search

\\u000a Artificial prostheses that are made of biomaterials hold great promise in nerve tissue engineering for the regeneration of\\u000a nervous system after injury. Silk fibroin, a fibrous protein from the domesticated silkworm, Bombyx mori, has been studied\\u000a extensively for its biocompatibility with a variety of cells. However, little is known about the tussah silk fibroin (TSF),\\u000a a type of wild silks

J. Qu; L. Xin; X. Xu; F. Zhang; B. Zuo; H. Zhang

148

Spider Webs and Silks.  

ERIC Educational Resources Information Center

|Compares the attributes of the silk from spiders with those of the commercially harvested silk from silkworms. Discusses the evolution, design, and effectiveness of spider webs; the functional mechanics of the varieties of silk that can be produced by the same spider; and the composite, as well as molecular, structure of spider silk thread. (JJK)|

Vollrath, Fritz

1992-01-01

149

Sequence Conservation in the C-terminal Region of Spider Silk Proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (A.raneidae)  

Microsoft Academic Search

The polymerase chain reaction (PCR) has been used to amplify the portion of the Spidroin 1 gene that codes for the C-terminal part of the silk protein of the spider NephiZa clavipes. Along with some substitution muta- tions of minor consequence, the PCR-derived sequence reveals an additional base missing from the previously published NephiZa Spidroin 1 sequence. Comparison of the

Richard BeckwittS

150

A novel method for dissolution and stabilization of non-mulberry silk gland protein fibroin using anionic surfactant sodium dodecyl sulfate.  

PubMed

The importance of silk protein has increased because of its potential use as a natural biopolymer for tissue engineering and biomedical applications. In this report we show a novel and ecofriendly method for dissolution of gland silk protein fibroin. Non-mulberry silk fibroin from mature fifth instar larvae of Antheraea mylitta was found to be optimally soluble in 1% (w/v) anionic surfactant sodium dodecyl sulfate (SDS). Regenerated fibroin showed distinct bands of approximately 395 and 197 kDa on electrophoresis in non-reducing and reducing conditions, respectively. Enhanced fibroin dissolution via internalization of hydrophobic amino groups inside a hydrophilic amino acid core in the form of micelles was observed. Prolonged storage stability without gelation of SDS-extracted fibroin was seen. Atomic force microscopy showed micellar aggregation with mean micellar aggregation size of 8 nm. Circular dichroism spectroscopy revealed predominantly helical conformation due to surfactant addition with internal protein conformational changes as revealed by fluorescence spectroscopic studies. PMID:17969177

Mandal, Biman B; Kundu, S C

2008-04-15

151

Silk film biomaterials for ocular surface repair  

NASA Astrophysics Data System (ADS)

Current biomaterial approaches for repairing the cornea's ocular surface upon injury are partially effective due to inherent material limitations. As a result there is a need to expand the biomaterial options available for use in the eye, which in turn will help to expand new clinical innovations and technology development. The studies illustrated here are a collection of work to further characterize silk film biomaterials for use on the ocular surface. Silk films were produced from regenerated fibroin protein solution derived from the Bombyx mori silkworm cocoon. Methods of silk film processing and production were developed to produce consistent biomaterials for in vitro and in vivo evaluation. A wide range of experiments was undertaken that spanned from in vitro silk film material characterization to in vivo evaluation. It was found that a variety of silk film properties could be controlled through a water-annealing process. Silk films were then generated that could be use in vitro to produce stratified corneal epithelial cell sheets comparable to tissue grown on the clinical standard substrate of amniotic membrane. This understanding was translated to produce a silk film design that enhanced corneal healing in vivo on a rabbit injury model. Further work produced silk films with varying surface topographies that were used as a simplified analog to the corneal basement membrane surface in vitro. These studies demonstrated that silk film surface topography is capable of directing corneal epithelial cell attachment, growth, and migration response. Most notably epithelial tissue development was controllably directed by the presence of the silk surface topography through increasing cell sheet migration efficiency at the individual cellular level. Taken together, the presented findings represent a comprehensive characterization of silk film biomaterials for use in ocular surface reconstruction, and indicate their utility as a potential material choice in the development of innovative procedures and technologies for corneal repair.

Lawrence, Brian David

152

Silk layering as studied with neutron reflectivity.  

PubMed

Neutron reflectivity (NR) measurements of ultrathin surface films (below 30 nm) composed of Bombyx mori silk fibroin protein in combination with atomic force microscopy and ellipsometry were used to reveal the internal structural organization in both dry and swollen states. Reconstituted aqueous silk solution deposited on a silicon substrate using the spin-assisted layer-by-layer (SA-LbL) technique resulted in a monolayer silk film composed of random nanofibrils with constant scattering length density (SLD). However, a vertically segregated ordering with two different regions has been observed in dry, thicker, seven-layer SA-LbL silk films. The vertical segregation of silk multilayer films indicates the presence of a different secondary structure of silk in direct contact with the silicon oxide surface (first 6 nm). The layered structure can be attributed to interfacial ?-sheet crystallization and the formation of well-developed nanofibrillar nanoporous morphology for the initially deposited silk surface layers with the preservation of less dense, random coil secondary structure for the layers that follow. This segregated structure of solid silk films defines their complex nonuniform behavior in the D(2)O environment with thicker silk films undergoing delamination during swelling. For a silk monolayer with an initial thickness of 6 nm, we observed the increase in the effective thickness by 60% combined with surprising decrease in density. Considering the nanoporous morphology of the hydrophobic silk layer, we suggested that the apparent increase in its thickness in liquid environment is caused by the air nanobubble trapping phenomenon at the liquid-solid interface. PMID:22697306

Wallet, Brett; Kharlampieva, Eugenia; Campbell-Proszowska, Katie; Kozlovskaya, Veronika; Malak, Sidney; Ankner, John F; Kaplan, David L; Tsukruk, Vladimir V

2012-07-26

153

Synchrotron FTIR microspectroscopy of single natural silk fibers.  

PubMed

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 ?-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 ?-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. PMID:21790142

Ling, Shengjie; Qi, Zeming; Knight, David P; Shao, Zhengzhong; Chen, Xin

2011-08-04

154

Pulsed laser deposition of silk protein: Effect of photosensitized-ablation on the secondary structure in thin deposited films  

NASA Astrophysics Data System (ADS)

Silk fibroin is a simple protein expected to have functional applications in medicine and bioelectronics. The primary structure of this protein is quite simple, and the main secondary structures are ?-sheet crystals and amorphous random coils. In the present study, we investigated pulsed laser deposition (PLD) of fibroin with the ?-sheet structures as targets. The primary and secondary structures in films deposited were analyzed using infrared spectroscopy. Normal laser deposition at 351 nm using neat fibroin targets produced thin films of fibroin with a random coiled structure. Ablation was triggered by two-photonic excitation of the peptide chains, which resulted in the destruction of ?-sheet structure in PLD. In order to avoid the two-photonic excitation, we adopted a PLD method utilizing anthracene (5-0.1 wt %) in a photosensitized reaction involving doped fibroin targets. Laser light (351 or 355 nm) was absorbed only by anthracene, which plays an important role converting photon energy to thermal energy with great ablation efficiency. Thin fibroin films deposited by this method had both random coil and ?-sheet structures. As the dopant concentration and laser fluence decreased, the ratio of ?-sheet domain to random coil increased in thin deposited films.

Tsuboi, Yasuyuki; Goto, Masaharu; Itaya, Akira

2001-06-01

155

Molecular Mechanism of Spider Silk Elasticity. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

Spider major ampullate (drag-line) silk is an extracellular fibrous protein which has impressive characteristics of strength and elasticity. This silk has been hypothesized to predominantly consist of a single protein, containing regions of antiparallel B...

Z. Dong R. V. Lewis C. R. Middaugh

1991-01-01

156

Transparent and flexible resistive switching memory devices with a very high ON/OFF ratio using gold nanoparticles embedded in a silk protein matrix.  

PubMed

The growing demand for biomaterials for electrical and optical devices is motivated by the need to make building blocks for the next generation of printable bio-electronic devices. In this study, transparent and flexible resistive memory devices with a very high ON/OFF ratio incorporating gold nanoparticles into the Bombyx mori silk protein fibroin biopolymer are demonstrated. The novel electronic memory effect is based on filamentary switching, which leads to the occurrence of bistable states with an ON=OFF ratio larger than six orders of magnitude. The mechanism of this process is attributed to the formation of conductive filaments through silk fibroin and gold nanoparticles in the nanocomposite. The proposed hybrid bio-inorganic devices show promise for use in future flexible and transparent nanoelectronic systems. PMID:23912245

Gogurla, Narendar; Mondal, Suvra P; Sinha, Arun K; Katiyar, Ajit K; Banerjee, Writam; Kundu, Subhas C; Ray, Samit K

2013-08-30

157

The Thr- and Ala-rich hyperactive antifreeze protein from inchworm folds as a flat silk-like ?-helix.  

PubMed

Inchworm larvae of the pale beauty geometer moth, Campaea perlata, exhibit strong (6.4 °C) freezing point depression activity, indicating the presence of hyperactive antifreeze proteins (AFPs). We have purified two novel Thr- and Ala-rich AFPs from the larvae as small (?3.5 kDa) and large (?8.3 kDa) variants and have cloned the cDNA sequences encoding both. They have no homology to known sequences in current BLAST databases. However, these proteins and the newly characterized AFP from the Rhagium inquisitor beetle both contain stretches rich in alternating Thr and Ala residues. On the basis of these repeats, as well as the discontinuities between them, a detailed structural model is proposed for the 8.3 kDa variant. This 88-residue protein is organized into an extended parallel-stranded ?-helix with seven strands connected by classic ?-turns. The alternating ?-strands form two ?-sheets with a thin core composed of interdigitating Ala and Ser residues, similar to the thin hydrophobic core proposed for some silks. The putative ice-binding face of the protein has a 4 × 5 regular array of Thr residues and is remarkably flat. In this regard, it resembles the nonhomologous Thr-rich AFPs from other moths and some beetles, which contain two longer rows of Thr in contrast to the five shorter rows in the inchworm protein. Like that of some other hyperactive AFPs, the spacing between these ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice. PMID:21486083

Lin, Feng-Hsu; Davies, Peter L; Graham, Laurie A

2011-05-04

158

Consequences of forced silking.  

PubMed

The forced silking of a spider to obtain major ampullate (MA) silk for experiments is a standard practice; however, this method may have profound effects on the resulting silk's properties. Experiments were performed to determine the magnitude of the difference in the forces required to draw silk from the MA gland between unrestrained spiders descending on their draglines and restrained spiders from which MA silk was drawn with a motor. The results show that freely falling spiders can spool silk with as little as 0.1 body weights of force, which generates a stress that is about 2% of the silk's tensile strength. In contrast, forcibly silked spiders apply as much as 4 body weights of force with an internal braking mechanism, and this force creates silk stresses in excess of 50% of the silk's tensile strength. The large forces observed in forced silking should strongly affect the draw alignment of the polymer network in the newly spun fibers, and this may account for the differences in material properties observed between naturally spun and forcibly spun MA silks. In addition, the heat produced by the internal friction brake during forced silking may set the upper limit of forced silking speed. PMID:15132653

Ortlepp, Christine S; Gosline, John M

159

Statistical approaches for investigating silk properties  

NASA Astrophysics Data System (ADS)

Amino acid repeats or motifs have engendered interest because of their significance for protein physical characteristics as well as folding properties. Spider dragline silk proteins are unique because they are composed of long repetitive sections and relatively short non-repetitive sections that are known to interact to generate the very peculiar mechanical and solubility properties of silk. Computational analysis compared with in vitro measurements suggest that the silks achieve their unique pattern of extreme solubility inside the spider glands/complete insolubility outside by correlating their repetitive hydrophobic regions through a type of stochastic resonance, generated by the addition of the non-repetitive sequences to a basically periodic hydrophobicity pattern.

Zbilut, J. P.; Scheibel, T.; Huemmerich, D.; Webber, C. L., Jr.; Colafranceschi, M.; Giuliani, A.

2006-02-01

160

Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation  

Microsoft Academic Search

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and ‘inverse transition cycling’. Laboratory

Jürgen Scheller; Daniele Henggeler; Angelika Viviani; Udo Conrad

2004-01-01

161

Controlling silk fibroin particle features for drug delivery  

PubMed Central

Silk proteins are a promising material for drug delivery due to their aqueous processability, biocompatibility, and biodegradability. A simple aqueous preparation method for silk fibroin particles with controllable size, secondary structure and zeta potential is reported. The particles were produced by salting out a silk fibroin solution with potassium phosphate. The effect of ionic strength and pH of potassium phosphate solution on the yield and morphology of the particles was determined. Secondary structure and zeta potential of the silk particles could be controlled by pH. Particles produced by salting out with 1.25 M potassium phosphate pH 6 showed a dominating silk II (crystalline) structure whereas particles produced at pH 9 were mainly composed of silk I (less crystalline). The results show that silk I rich particles possess chemical and physical stability and secondary structure which remained unchanged during post treatments even upon exposure to 100% ethanol or methanol. A model is presented to explain the process of particle formation based on intra- and intermolecular interactions of the silk domains, influenced by pH and kosmotrope salts. The reported silk fibroin particles can be loaded with small molecule model drugs, such as alcian blue, rhodamine B, and crystal violet, by simple absorption based on electrostatic interactions. In vitro release of these compounds from the silk particles depends on charge – charge interactions between the compounds and the silk. With crystal violet we demonstrated that the release kinetics are dependent on the secondary structure of the particles.

Lammel, Andreas; Hu, Xiao; Park, Sang-Hyug; Kaplan, David L.; Scheibel, Thomas

2010-01-01

162

Surface properties and conformation of Nephila clavipes spider recombinant silk proteins at the air-water interface.  

PubMed

The dragline fiber of spiders is composed of two proteins, the major ampullate spidroins I and II (MaSpI and MaSpII). To better understand the assembly mechanism and the properties of these proteins, the adsorption behavior of the recombinant proteins of the spider Nephila clavipes produced by Nexia Biotechnologies Inc. has been studied at the air-water interface using ellipsometry, surface pressure, rheological, and infrared measurements. The results show that the adsorption is more rapid and more molecules are present at the interface for MaSpII than for MaSpI. MaSpII has thus a higher affinity for the interface than MaSpI, which is consistent with its higher aggregation propensity in water. The films formed at the interface consist of networks containing a high content of intermolecular beta-sheets as revealed by the in situ polarization modulation infrared absorption reflection spectra. The infrared results further demonstrate that, for MaSpI, the beta-sheets are formed as soon as the proteins adsorb to the interface while for MaSpII the beta-sheet formation occurs more slowly. The amount of beta-sheets is lower for MaSpII than for MaSpI, most likely due to the presence of proline residues in its sequence. Both proteins form elastic films, but they are heterogeneous for MaSpI and homogeneous for MaSpII most probably as a result of a more ordered and slower aggregation process for MaSpII. This difference in their mechanism of assembly and interfacial behaviors does not seem to arise from their overall hydrophobicity or from a specific pattern of hydrophobicity, but rather from the longer polyalanine motifs, lower glycine content, and higher proline content of MaSpII. The propensity of both spidroins to form beta-sheets, especially the polyalanine blocks, suggests the participation of both proteins in the silk's beta-sheet crystallites. PMID:19400566

Renault, Anne; Rioux-Dubé, Jean-François; Lefèvre, Thierry; Pezennec, Stéphane; Beaufils, Sylvie; Vié, Véronique; Tremblay, Mélanie; Pézolet, Michel

2009-07-21

163

Exploiting spiders’ silk  

Microsoft Academic Search

Scientists and engineers have long envied spiders’ ability to manufacture a material that is simultaneously strong, fine, and tough. This combination of properties makes spider silk an extremely attractive candidate for numerous applications in medicine, defense, and the leisure industry. Research teams worldwide are gradually piecing together the relationship between the silk’s make-up and its unusual — but highly useful

Paula Gould

2002-01-01

164

Extreme Diversity, Conservation, and Convergence of Spider Silk Fibroin Sequences  

NASA Astrophysics Data System (ADS)

Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million years of evolutionary history. The retention of these conserved motifs since the Mesozoic and their convergent evolution in other structural superproteins imply that these sequences are central to understanding the exceptional mechanical properties of orb weaver silks.

Gatesy, John; Hayashi, Cheryl; Motriuk, Dagmara; Woods, Justin; Lewis, Randolph

2001-03-01

165

Extreme diversity, conservation, and convergence of spider silk fibroin sequences.  

PubMed

Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million years of evolutionary history. The retention of these conserved motifs since the Mesozoic and their convergent evolution in other structural superproteins imply that these sequences are central to understanding the exceptional mechanical properties of orb weaver silks. PMID:11283372

Gatesy, J; Hayashi, C; Motriuk, D; Woods, J; Lewis, R

2001-03-30

166

Proteomic profiling of the photo-oxidation of silk fibroin: implications for historic tin-weighted silk.  

PubMed

The stability of silk proteins to ultraviolet light is an issue of significant concern in both the appearance retention of silk-derived products and the preservation of historic silk textiles. Until now, evaluation of silk degradation has only been performed at the holistic, rather than molecular level. This article describes the first proteomic profiling of silk photo-oxidation, characterizing protein primary level modification leading to coloration changes, and evaluating the effects of tin weighting on photodegradation. Heavy-chain fibroin, the main proteinaceous component of the silk thread, is a repetitive, highly crystalline protein with a content rich in tyrosine. Photoproducts of tyrosine were characterized and the levels of oxidative modification at the protein primary structural level correlated with changes in coloration and tensile strength. The effect of tin as a weighting agent used on historical fabrics was examined. Tin-weighted fabrics were evaluated following two treatments (pink and dynamite) and proteomic analysis revealed a significant increase in oxidatively modified amino acid residues within the pink-treated silk. These findings offer new insight into the molecular-level oxidation of silk proteins under UV exposure, and the effects of silk treatments in either exacerbating or ameliorating this degradation. PMID:22554154

Solazzo, Caroline; Dyer, Jolon M; Deb-Choudhury, Santanu; Clerens, Stefan; Wyeth, Paul

2012-05-23

167

Conservation of essential design features in coiled coil silks.  

PubMed

Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended beta-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small ( approximately 30 kDa each) and nonrepetitive and adopt a coiled coil structure. We examined silks from the 3 superfamilies of the Aculeata (Hymenoptera: Apocrita) by infrared spectroscopy and found coiled coil structure in bees (Apoidea) and in ants (Vespoidea) but not in parasitic wasps of the Chrysidoidea. We subsequently identified and sequenced the silk genes of bumblebees, bulldog ants, and weaver ants and compared these with honeybee silk genes. Each species produced orthologues of the 4 small fibroin proteins identified in honeybee silk. Each fibroin contained a continuous predicted coiled coil region of around 210 residues, flanked by 23-160 residue length N- and C-termini. The cores of the coiled coils were unusually rich in alanine. There was extensive sequence divergence among the bee and ant silk genes (<50% similarity between the alignable regions of bee and ant sequences), consistent with constant and equivalent divergence since the bee/ant split (estimated to be 155 Myr). Despite a high background level of sequence diversity, we have identified conserved design elements that we propose are essential to the assembly and function of coiled coil silks. PMID:17703050

Sutherland, Tara D; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Trueman, John W H; Haritos, Victoria S

2007-08-16

168

Continuous production of flexible fibers from transgenically produced honeybee silk proteins.  

PubMed

Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting. PMID:23881528

Poole, Jacinta; Church, Jeffrey S; Woodhead, Andrea L; Huson, Mickey G; Sriskantha, Alagacone; Kyratzis, Ilias L; Sutherland, Tara D

2013-07-23

169

Carotenoid Silk Coloration Is Controlled by a Carotenoid-Binding Protein, a Product of the Yellow blood Gene  

Microsoft Academic Search

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular

Takashi Sakudoh; Hideki Sezutsu; Takeharu Nakashima; Isao Kobayashi; Hirofumi Fujimoto; Keiro Uchino; Yutaka Banno; Hidetoshi Iwano; Hideaki Maekawa; Toshiki Tamura; Hiroshi Kataoka; Kozo Tsuchida

2007-01-01

170

Amino Acid Composition-Dependent Elasticity of SPIDER Silk  

NASA Astrophysics Data System (ADS)

Spider silk exhibits excellent mechanical features in both toughness and extensibility. In recent years considerable investigations have focused on it. The understanding of spider silk protein is important for the development bionic silk. In this paper, we study by Monte Carlo simulation the force-of-extension property of spider silk proteins as a function of the residue composition for major and minor ampullate glands of typical Araneid orb weaver as well as that for one artificial spider silk. The results are also compared with those from a designed protein whose amino acid composition is uniform. The results clearly show that the major and minor ampullate gland proteins are much tougher than the designed protein, whereas the artificial protein, as a model of a nature spider silk, does have good mechanical properties. Our simulation reveals that mechanical property of a spider silk protein is dependent on its amino acid composition and that an excellent result of natural evolution is manifest in the composition of the spider silk protein.

Zhang, Yong; Dai, Luru; Ou-Yang, Zhong-Can

171

In vivo biological responses to silk proteins functionalized with bone sialoprotein.  

PubMed

Recombinant 6mer?+?BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer?+?BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer?+?BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications. PMID:23359587

Gomes, Sílvia; Gallego-Llamas, Jabier; Leonor, Isabel B; Mano, João F; Reis, Rui L; Kaplan, David L

2013-01-28

172

Silken toolkits: biomechanics of silk fibers spun by the orb web spider Argiope argentata (Fabricius 1775).  

PubMed

Orb-weaving spiders spin five fibrous silks from differentiated glands that contain unique sets of proteins. Despite diverse ecological functions, the mechanical properties of most of these silks are not well characterized. Here, we quantify the mechanical performance of this toolkit of silks for the silver garden spider Argiope argentata. Four silks exhibit viscoelastic behaviour typical of polymers, but differ statistically from each other by up to 250% in performance, giving each silk a distinctive suite of material properties. Major ampullate silk is 50% stronger than other fibers, but also less extensible. Aciniform silk is almost twice as tough as other silks because of high strength and extensibility. Capture spiral silk, coated with aqueous glue, is an order of magnitude stretchier than other silks. Dynamic mechanical properties are qualitatively similar, but quantitatively vary by up to 300% among silks. Storage moduli are initially nearly constant and increase after fiber yield, whereas loss tangents reach maxima of 0.1-0.2 at the yield. The remarkable mechanical diversity of Argiope argentata silks probably results in part from the different molecular structures of fibers and can be related to the specific ecological role of each silk. Our study indicates substantial potential to customize the mechanics of bioengineered silks. PMID:16788028

Blackledge, Todd A; Hayashi, Cheryl Y

2006-07-01

173

Designing Spider Silk Genes for Materials Applications.  

National Technical Information Service (NTIS)

This project is designed to create new proteins based on naturally occurring spider silk proteins with the goal of controlling elasticity and tensile strength in fibers spun from the proteins. The new genes have been constructed in year 1, the proteins ha...

R. V. Lewis

2006-01-01

174

Materials Fabrication from Bombyx mori Silk Fibroin  

PubMed Central

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years, and it can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent processing methods can be used to generate silk biomaterials for a range of applications. In this protocol we include methods to extract silk from B. mori cocoons in order to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, and for drug delivery.

Rockwood, Danielle N.; Preda, Rucsanda C.; Yucel, Tuna; Wang, Xiaoqin; Lovett, Michael L.; Kaplan, David L.

2013-01-01

175

Synthesis and characterization of water-soluble silk peptides and recombinant silk protein containing polyalanine, the integrin binding site, and two glutamic acids at each terminal site as a possible candidate for use in bone repair materials.  

PubMed

The recombinant proteins [EE(A)12EETGRGDSPAAS]n (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E2(A)nE2TGRGDSPAE2(A)nE2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO(-) to COOH of the E2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)12EETGRGDSPAAS]n (n = 5,10) by expression in Escherichia coli . The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)12EETGRGDSPAAS]10 has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material. PMID:24004453

Asakura, Tetsuo; Suzuki, Yu; Nagano, Aya; Knight, David; Kamiya, Masakatsu; Demura, Makoto

2013-09-19

176

Variation in the material properties of spider dragline silk across species  

Microsoft Academic Search

Spiders produce high performance fibers that compare favorably with the best manmade fibers in strength and toughness. The amino acid sequences of silk proteins have been determined for a number of silk types and species, revealing extensive variation. This variation in sequence is hypothesized to confer different material properties. However, the material properties of silk have been characterized from only

B. O. Swanson; T. A. Blackledge; J. Beltrán; C. Y. Hayashi

2006-01-01

177

Multiple Recombining Loci Encode MaSp1, the Primary Constituent of Dragline Silk, in Widow Spiders (Latrodectus: Theridiidae)  

Microsoft Academic Search

Spiders spin a functionally diverse array of silk fibers, each composed of one or more unique proteins. Most of these proteins, in turn, are encoded by members of a single gene family thought to have arisen through duplication and divergence of an ancestral silk gene. Because of its remarkable mechanical properties, orb weaver dragline silk, a composite of 2 proteins

Nadia A. Ayoub; Cheryl Y. Hayashi

2008-01-01

178

Water permeability of spider dragline silk.  

PubMed

The water permeability of spider dragline silk was studied by measuring changes in amide deuteration of D(2)O-soaked silk with solid-state NMR. (13)C-D rotational-echo double-resonance (REDOR) NMR experiments showed that chemical exchange of amide hydrogen occurs in a large fraction of amino acids, including over 50% of alanine residues, which are known to exist predominantly in beta-sheet crystallites. This suggests that a substantial fraction of the crystalline regions are permeable to water, at least on the time scale of hours, implying that they are more dynamic, and therefore susceptible to chemical exchange with water, than previously thought. Wideline deuterium NMR spectra of dried D(2)O-soaked silk showed a combination of quadrupolar broadened and motionally averaged isotropic components whose intensities change on the time scale of hours. These results are interpreted in terms of chemical exchange between deuterium on the protein backbone, residual water within the silk, and water vapor in the ambient atmosphere. A simple compartmental model fits the results well and yields rate constants for the exchange processes. The model requires the inclusion of a compartment that does not undergo exchange. This compartment, likely related to the crystalline region, is interesting because it is accessible to water in wet silk, but impervious to any remaining free water when the silk is dried. PMID:19331319

Li, Xiang; Eles, Philip T; Michal, Carl A

2009-05-11

179

Physical characterization of functionalized spider silk: electronic and sensing properties  

Microsoft Academic Search

This work explores functional, fundamental and applied aspects of naturally harvested spider silk fibers. Natural silk is a protein polymer where different amino acids control the physical properties of fibroin bundles, producing, for example, combinations of beta-sheet (crystalline) and amorphous (helical) structural regions. This complexity presents opportunities for functional modification to obtain new types of material properties. Electrical conductivity is

Eden Steven; Jin Gyu Park; Anant Paravastu; Elsa Branco Lopes; James S. Brooks; Ongi Englander; Theo Siegrist; Papatya Kaner; Rufina G. Alamo

2011-01-01

180

Water-Insoluble Silk Films with Silk I Structure  

PubMed Central

Water-insoluble regenerated silk materials are normally achieved by increasing ?-sheet content (silk II). In the present study, water-insoluble silk films were prepared by controlling very slow drying of B. mori silk solutions, resulting in the formation of stable films with dominating silk I instead of silk II structure. Wide angle x-ray scattering (WAXS) indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water- and methanol-annealed silk films had a higher silk II content. The silk films prepared through slow drying had a globule-like structure in the core with nano-filaments. The core region was composed of silk I and silk II, and these regions are surrounded by hydrophilic nano-filaments containing random, turns, and ?-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. DSC results revealed that silk I crystals had stable thermal properties up to 250°C, without crystallization above the Tg, but degraded in lower temperature than silk II structure. Compared with water- and methanol-annealed films, the films prepared through slow drying achieved better mechanical ductility and more rapid enzymatic degradation, reflective of the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated in the present approach of very slow drying, mimicking the natural process. The results also point to a new mode to generate new types of silk biomaterials, where mechanical properties can be enhanced, and degradation rates increased, yet water insolubility is maintained along with low beta sheet content.

Lu, Qiang; Hu, Xiao; Wang, Xiaoqin; Kluge, Jonathan A.; Lu, Shenzhou; Cebe, Peggy; Kaplan, David L.

2009-01-01

181

Sequential origin in the high performance properties of orb spider dragline silk.  

PubMed

Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376?MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230?MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers. PMID:23110251

Blackledge, Todd A; Pérez-Rigueiro, José; Plaza, Gustavo R; Perea, Belén; Navarro, Andrés; Guinea, Gustavo V; Elices, Manuel

2012-10-29

182

Sequential origin in the high performance properties of orb spider dragline silk  

NASA Astrophysics Data System (ADS)

Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376 MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230 MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.

Blackledge, Todd A.; Pérez-Rigueiro, José; Plaza, Gustavo R.; Perea, Belén; Navarro, Andrés; Guinea, Gustavo V.; Elices, Manuel

2012-10-01

183

Sequential origin in the high performance properties of orb spider dragline silk  

PubMed Central

Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376?MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230?MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.

Blackledge, Todd A.; Perez-Rigueiro, Jose; Plaza, Gustavo R.; Perea, Belen; Navarro, Andres; Guinea, Gustavo V.; Elices, Manuel

2012-01-01

184

Silk Fibroin Encapsulated Powder Reservoirs for Sustained Release of Adenosine  

PubMed Central

Due to its unique properties, silk fibroin was studied as a biodegradable polymer vehicle for sustained, local delivery of the anticonvulsant adenosine from encapsulated reservoirs. Silk is a biologically derived protein polymer that is biocompatible, mechanically strong and degrades to non-toxic products in vivo. To achieve local, sustained, controlled adenosine release from fully degradable implants, solid adenosine powder reservoirs were coated with silk fibroin. Material properties of the silk coating including thickness, crystallinity and morphology were investigated to assess the relationships between silk coating biomaterial features and adenosine release from silk encapsulated reservoirs. Reservoir coating thickness was varied through manipulation of the silk coating solution concentration and number of coatings applied. Release studies were also performed in proteinase type XIV to model the effects of degradation. Increasing the barrier to diffusion, either by increasing coating thickness or crystallinity was found to delay adenosine burst, decrease average daily release rate, and increase duration of release. In the case of encapsulated reservoirs coated with eight layers of 8% (w/v) silk, a linear release profile was observed and adenosine release was sustained for 14 days. The ability to achieve nearly constant release for two weeks for adenosine via control of the silk coating suggests these encapsulated reservoirs represent a novel system for delivering adenosine. We anticipate that this approach could also be extended to other implant needs and small molecule drugs to treat a range of clinical needs.

Pritchard, Eleanor M.; Szybala, Cory; Boison, Detlev; Kaplan, David L.

2010-01-01

185

Silk Batik using Cochineal Dye  

Technology Transfer Automated Retrieval System (TEKTRAN)

The history of silk, including sericulture (the production of raw silk, which requires the raising of silkworms on their natural diet, mulberry leaves) and silk manufacturing, is rich and extensive. It encompasses several famous “silk roads” (trade routes), various cultures and technologies, ideas,...

186

Antimicrobial functionalized genetically engineered spider silk  

PubMed Central

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.

Gomes, Silvia; Leonor, Isabel B.; Mano, Joao F.; Reis, Rui L.; Kaplan, David L.

2011-01-01

187

Art on Silk Hoops  

ERIC Educational Resources Information Center

|Painting on silk has a magic all its own. Versions of painting on silk can be found throughout the world from Japan and Europe to the United States. Themes for the paintings can be most any type of design or imagery. Applying the liquid dyes is exciting, as the vivid liquid colors flow and blend into the fabric. The process captures students'…

Padrick, Deborah

2012-01-01

188

Silk Road Seattle  

NSDL National Science Digital Library

Silk Road Seattle is a "collaborative public education project using the 'Silk Road' theme to explore cultural interaction across Eurasia from the beginning of the Common Era (A.D.) to the Sixteenth Century." The Silk Road is a term understood to have been the overland trade route from China to the Mediterranean, opened first in the 2nd century and coming to an end between the 15th and 17th centuries. Traditional discussions of the Silk Road, however, recognize that there were branches that went into South Asia, or extended from Central Asia north of the Caspian Sea to the Black Sea. Sponsored primarily by the Simpson Center for the Humanities at the University of Washington and directed by Professors Daniel C. Waugh, Joel Walker, and Cynthea Bogel, this Web site contains a vast range of Silk Road materials. These materials include a list of texts that may be used in teaching and learning about the Silk Road, a section on cities and architecture along the Silk Road, information on traditional culture in Central Asia, a page of teaching and learning guides that list and annotate Silk Road materials, and a virtual art exhibit. This site is still in a constructive stage; therefore, every link is not yet accessible. In short, this site may be of value for a range of audiences, including school children, teachers, college and graduate students, and independent adult learners.

2001-01-01

189

Silk material modification and microfabrication strategies for applications in biosensors and drug delivery  

NASA Astrophysics Data System (ADS)

Silk has proven to be a promising biomaterial for the development of a novel generation of biomedical devices due to the material's intrinsic properties of biocompatibility and biodegradability. To further the development of silk-based devices, augmented functionality can be provided to silk by means of microfabrication and material functionalization. In this dissertation, we set out to explore possibilities of silk-based biomedical device development with particular attention to different fabrication strategies that can be leveraged for this purpose, taking inspiration from conventional lithography, contact imprinting and chemical modification of the silk biomaterial. In particular, we have produced a novel silk-based drug delivery device, in the form of microneedles. For this purpose we have developed micromolding strategies which allow the fabrication of high aspect ratio silk structures. Furthermore, we have produced a THz split ring resonator based sensor device on silk films. To realize this device, we have developed a convenient fabrication method, allowing transfer of previously microfabricated metal and oxide microstructures to the silk film surface. This method has proven useful for hard masking and patterning silk films with reactive ion etching. Moreover, we have explored patterning semiconductor and glass substrates with silk films. For this purpose we have modified a standard microfabrication method -lift off- to be amendable to silk. Furthermore, combining silk based fabrication and material functionalization has lead to microfluidics pH sensing applications. Finally we have explored the possibility of utilizing silk for injectable and biodegradable glucose sensors. For this purpose, we have doped silk hydrogels with a fluorescence based protein biosensor. The advanced silk fabrication and material synthesis strategies and the resulting novel devices presented here could potentially lead to a new class of biomedical applications.

Tsioris, Konstantinos

190

Unraveling A Trap-and-Trigger Mechanism in the pH-Sensitive Self-Assembly of Spider Silk Proteins.  

PubMed

When the major ampulate spidroins (MaSp1) are called upon to form spider dragline silk, one of nature's most amazing materials, a small drop in pH must occur. Using a state-of-the-art simulation technique, constant pH molecular dynamics, we discovered a few residues that respond to the pH signal in the dimerization of the N-terminal domain (NTD) of MaSp1 which is an integral step in the fiber assembly. At neutral pH the deprotonation of Glu79 and Glu119 leads to water penetration and structural changes at the monomer-monomer binding interface. At strongly acidic pH, the protonation of Asp39 and Asp40 weakens the electrostatic attraction between the monomers. Thus, we propose a "trap-and-trigger" mechanism whereby the intermolecular salt-bridges at physiologically relevant pH conditions always act as a stabilizing "trap" favoring dimerization. As pH is lowered to about 6, Glu79 and Glu119 become protonated, triggering the dimerization and subsequent silk formation. We speculate that this type of mechanism is operative in many other pH-sensitive biological processes. PMID:22866209

Wallace, Jason A; Shen, Jana K

2012-02-15

191

Unraveling A Trap-and-Trigger Mechanism in the pH-Sensitive Self-Assembly of Spider Silk Proteins  

PubMed Central

When the major ampulate spidroins (MaSp1) are called upon to form spider dragline silk, one of nature’s most amazing materials, a small drop in pH must occur. Using a state-of-the-art simulation technique, constant pH molecular dynamics, we discovered a few residues that respond to the pH signal in the dimerization of the N-terminal domain (NTD) of MaSp1 which is an integral step in the fiber assembly. At neutral pH the deprotonation of Glu79 and Glu119 leads to water penetration and structural changes at the monomer-monomer binding interface. At strongly acidic pH, the protonation of Asp39 and Asp40 weakens the electrostatic attraction between the monomers. Thus, we propose a “trap-and-trigger” mechanism whereby the intermolecular salt-bridges at physiologically relevant pH conditions always act as a stabilizing “trap” favoring dimerization. As pH is lowered to about 6, Glu79 and Glu119 become protonated, triggering the dimerization and subsequent silk formation. We speculate that this type of mechanism is operative in many other pH-sensitive biological processes.

Wallace, Jason A.; Shen, Jana K.

2012-01-01

192

Carbondioxide gating in silk cocoon.  

PubMed

Silk is the generic name given to the fibrous proteins spun by a number of arthropods. During metamorphosis, the larva of the silk producing arthropods excrete silk-fiber from its mouth and spun it around the body to form a protective structure called cocoon. An adult moth emerges out from the cocoon after the dormant phase (pupal phase) varying from 2 weeks to 9 months. It is intriguing how CO(2)/O(2) and ambient temperature are regulated inside the cocoon during the development of the pupa. Here we show that the cocoon membrane is asymmetric, it allows preferential gating of CO(2) from inside to outside and it regulates a physiological temperature inside the cocoon irrespective of the surrounding environment temperature. We demonstrate that under simulating CO(2) rich external environment, the CO(2) does not diffuse inside the cocoon. Whereas, when CO(2) was injected inside the cocoon, it diffuses out in 20 s, indicating gating of CO(2) from inside to outside the membrane. Removal of the calcium oxalate hydrate crystals which are naturally present on the outer surface of the cocoon affected the complete blockade of CO(2) flow from outside to inside suggesting its role to trap most of the CO(2) as hydrogen bonded bicarbonate on the surface. The weaved silk of the cocoon worked as the second barrier to prevent residual CO(2) passage. Furthermore, we show that under two extreme natural temperature regime of 5 and 50 °C, a temperature of 25 and 34 °C respectively were maintained inside the cocoons. Our results demonstrate, how CO(2) gating and thermoregulation helps in maintaining an ambient atmosphere inside the cocoon for the growth of pupa. Such natural architectural control of gas and temperature regulation could be helpful in developing energy saving structures and gas filters. PMID:22791361

Roy, Manas; Meena, Sunil Kumar; Kusurkar, Tejas Sanjeev; Singh, Sushil Kumar; Sethy, Niroj Kumar; Bhargava, Kalpana; Sarkar, Sabyasachi; Das, Mainak

2012-07-12

193

A silk fibroin based hepatocarcinoma model and the assessment of the drug response in hyaluronan-binding protein 1 overexpressed HepG2 cells.  

PubMed

Microenvironment around tumor cells plays an important role in its malignancy or invasiveness. Hyaluronan (HA), a major component of extracellular matrix is found to be elevated in most of cancerous niche/microenvironment and performs regulatory role in the progression of tumors and metastasis. Overexpression of the hyaladherin, hyaluronan-binding protein 1 (HABP1) in the hepatocarcinoma cells (HepG2) termed as HepR21 leads to enhanced cell proliferation with increased HA 'pool' associated with HA 'cables' indicating elevated tumorous potential under 2D culture conditions. For in vitro experimentation, scaffold based three dimensional niche modeling may have greater acceptance than conventional 2D culture condition. Thus, we have examined the influence of intrinsic properties of non-mulberry tropical tasar silk fibroin on the HepR21 cells in order to develop a 3D hepatocarcinoma construction to act as model. The scaffold of tasar silk fibroin of Antheraea mylitta when efficiently loaded with transformed hepatocarcinoma cells, HepR21; exhibits enhanced adhesiveness, viability, metabolic activity, proliferation and enlarged cellular morphology in 3D compared to its parent cell line HepG2, supporting the earlier observation made in 2D system. In addition, formation of multicellular aggregates, the indicator of tumor progression is also revealed in silk based 3D culture conditions. Further, the use of 4-MU (a hyaluronan synthase inhibitor) on HepR21 cells reduces the HA level and downregulates the expression of growth promoting factors like pAKT and PKC; while upregulating the expression of the tumor suppressor p53. Thus, 4-MU efficiently reduces the tumor potency associated with increased HA pool as well as HA cables and the effect of 4-MU doubling up as an anticancer agent in 2D and 3D are also comparable. The in vitro 3D multicellular model demonstrates the insight of hepatocarcinoma progression and offers the predictability of cellular response to transfection efficacy, drug treatment and therapeutic intervention. PMID:24016853

Kundu, Banani; Saha, Paramita; Datta, Kasturi; Kundu, Subhas C

2013-09-07

194

Increased molecular mobility in humid silk fibers under tensile stress  

NASA Astrophysics Data System (ADS)

Silk fibers are semicrystalline nanocomposite protein fibers with an extraordinary mechanical toughness that changes with humidity. Diffusive or overdamped motion on a molecular level is absent in dry silkworm silk, but present in humid silk at ambient temperature. This microscopic diffusion distinctly depends on the externally applied macroscopic tensile force. Quasielastic and inelastic neutron-scattering data as a function of humidity and of tensile strain on humid silk fibers support the model that both the adsorbed water and parts of the amorphous polymers participate in diffusive motion and are affected by the tensile force. It is notable that the quasielastic linewidth of humid silk at 100% relative humidity increases significantly with the applied force. The effect of the tensile force is discussed in terms of an increasing alignment of the polymer chains in the amorphous fraction with increasing tensile stress which changes the geometrical restrictions of the diffusive motions.

Seydel, Tilo; Knoll, Wiebke; Greving, Imke; Dicko, Cedric; Koza, Michael M.; Krasnov, Igor; Müller, Martin

2011-01-01

195

An Unlikely Silk: The Composite Material of Green Lacewing Cocoons  

SciTech Connect

Spiders routinely produce multiple types of silk; however, common wisdom has held that insect species produce one type of silk each. This work reports that the green lacewing (Mallada signata, Neuroptera) produces two distinct classes of silk. We identified and sequenced the gene that encodes the major protein component of the larval lacewing cocoon silk and demonstrated that it is unrelated to the adult lacewing egg-stalk silk. The cocoon silk protein is 49 kDa in size and is alanine rich (>40%), and it contains an {alpha}-helical secondary structure. The final instar lacewing larvae spin protein fibers of {approx}2 {mu}m diameter to construct a loosely woven cocoon. In a second stage of cocoon construction, the insects lay down an inner wall of lipids that uses the fibers as a scaffold. We propose that the silk protein fibers provide the mechanical strength of the composite lacewing cocoon whereas the lipid layer provides a barrier to water loss during pupation.

Weisman, Sarah; Trueman, Holly E.; Mudie, Stephen T.; Church, Jeffrey S.; Sutherland, Tara D.; Haritos, Victoria S. (CSIRO/MSE); (CSIRO)

2009-01-15

196

Clay-Enriched Silk Biomaterials for Bone Formation  

PubMed Central

The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone specific uses. Clay montmorillonite (Cloisite ® Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared to silk films doped with sodium silicate as controls for support of human bone marrow derived mesenchymal stem cells (hMSCs) in osteogenic culture. The cells adhered and proliferated on the silk/clay composites over two weeks. Quantitative real-time RT-PCR analysis revealed increased transcript levels for alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col I) osteogenic markers in the cells cultured on the silk/clay films in comparison to the controls. Early evidence for bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that the silk/clay composite systems may be useful for further study toward bone regenerative needs.

Mieszawska, Aneta J.; Llamas, Jabier Gallego; Vaiana, Christopher A.; Kadakia, Madhavi P.; Naik, Rajesh R.; Kaplan, David L.

2011-01-01

197

Spider silks from plants - a challenge to create native-sized spidroins.  

PubMed

Silk threads from spiders exhibit extraordinary mechanical properties, such as superior toughness and elasticity. Spider silks consist of several different large repetitive proteins that act as the basic materials responsible for these outstanding features. The production of spider silk protein variants in plants opens up new horizons in the production and functional investigation that enable the use of spider silks in innovative material development, nanotechnology and biomedicine in the future. This review summarizes and discusses production of spider silk protein variants in plants, especially with regards to plant expression systems, purification strategies, and characteristics of spider silk variants. Furthermore, the challenge of producing native-sized recombinant spidroins in planta is outlined, presenting three different strategies for achieving these high repetitive proteins with the help of non-repetitive C-terminal domains, crosslinking transglutaminase, and self-linking inteins. The potential of these fascinating proteins in medicine is also highlighted. PMID:24092675

Hauptmann, Valeska; Weichert, Nicola; Rakhimova, Marziya; Conrad, Udo

2013-10-01

198

Silk fibroin processing and thrombogenic responses.  

PubMed

Silkworm-derived fibroin, which constitutes the core of the silk filament, is an attractive protein-polymer for biomedical applications. Fibroin can also be processed into a variety of 2-D and 3-D formats to match morphological and structural features to specific applications. The focus of the present research was to correlate the structure of silk fibroin-derived biomaterials with plasma protein adsorption, platelet activation and inflammatory cell (THP-1 cell line) adhesion and activation. The amino-acid composition of the two types of silk studied influenced the crystallinity of the films, hydrophobicity, surface roughness and biological interactions. Protein adsorption was lower on samples with the higher crystallinity and hydrophobicity, in particular the chemotactic factors (C3a, C5a, C3b), while other proteins such as fibrinogen were comparable in terms of adsorption. As a consequence, platelets and immune cells responded differently to the various films obtained by following different processing protocols and stabilized by different methods (methanol or water vapour) in terms of their adherence, activation, and the secretion of inflammatory mediators by monocytes. The data presented here demonstrate that bioactivity can be influenced by changing the chemistry, such as the source of silk protein, or by the specific process used in the preparation of the materials used to assess biological responses. PMID:19793445

Motta, Antonella; Maniglio, Devid; Migliaresi, Claudio; Kim, Hyeon-Joo; Wan, Xianyan; Hu, Xiao; Kaplan, David L

2009-01-01

199

Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies.  

PubMed

The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering ?-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications. PMID:21216004

Guziewicz, Nicholas; Best, Annie; Perez-Ramirez, Bernardo; Kaplan, David L

2011-01-08

200

Liquid crystalline spinning of spider silk  

Microsoft Academic Search

Spider silk has outstanding mechanical properties despite being spun at close to ambient temperatures and pressures using water as the solvent. The spider achieves this feat of benign fibre processing by judiciously controlling the folding and crystallization of the main protein constituents, and by adding auxiliary compounds, to create a composite material of defined hierarchical structure. Because the `spinning dope'

Fritz Vollrath; David P. Knight

2001-01-01

201

Liquid crystalline spinning of spider silk  

Microsoft Academic Search

Spider silk has outstanding mechanical properties despite being spun at close to ambient temperatures and pressures using water as the solvent. The spider achieves this feat of benign fibre processing by judiciously controlling the folding and crystallization of the main protein constituents, and by adding auxiliary compounds, to create a composite material of defined hierarchical structure. Because the 'spinning dope'

Fritz Vollrath; David P. Knight

2001-01-01

202

Greatly Increased Toughness of Infiltrated Spider Silk  

NASA Astrophysics Data System (ADS)

In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials. A desire to produce these biomimicking new materials has stimulated materials scientists, and diverse approaches have been attempted. In contrast, research to improve the mechanical properties of biomaterials themselves by direct metal incorporation into inner protein structures has rarely been tried because of the difficulty of developing a method that can infiltrate metals into biomaterials, resulting in a metal-incorporated protein matrix. We demonstrated that metals can be intentionally infiltrated into inner protein structures of biomaterials through multiple pulsed vapor-phase infiltration performed with equipment conventionally used for atomic layer deposition (ALD). We infiltrated zinc (Zn), titanium (Ti), or aluminum (Al), combined with water from corresponding ALD precursors, into spider dragline silks and observed greatly improved toughness of the resulting silks. The presence of the infiltrated metals such as Al or Ti was verified by energy-dispersive x-ray (EDX) and nuclear magnetic resonance spectra measured inside the treated silks. This result of enhanced toughness of spider silk could potentially serve as a model for a more general approach to enhance the strength and toughness of other biomaterials.

Lee, Seung-Mo; Pippel, Eckhard; Gösele, Ulrich; Dresbach, Christian; Qin, Yong; Chandran, C. Vinod; Bräuniger, Thomas; Hause, Gerd; Knez, Mato

2009-04-01

203

Greatly increased toughness of infiltrated spider silk.  

PubMed

In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials. A desire to produce these biomimicking new materials has stimulated materials scientists, and diverse approaches have been attempted. In contrast, research to improve the mechanical properties of biomaterials themselves by direct metal incorporation into inner protein structures has rarely been tried because of the difficulty of developing a method that can infiltrate metals into biomaterials, resulting in a metal-incorporated protein matrix. We demonstrated that metals can be intentionally infiltrated into inner protein structures of biomaterials through multiple pulsed vapor-phase infiltration performed with equipment conventionally used for atomic layer deposition (ALD). We infiltrated zinc (Zn), titanium (Ti), or aluminum (Al), combined with water from corresponding ALD precursors, into spider dragline silks and observed greatly improved toughness of the resulting silks. The presence of the infiltrated metals such as Al or Ti was verified by energy-dispersive x-ray (EDX) and nuclear magnetic resonance spectra measured inside the treated silks. This result of enhanced toughness of spider silk could potentially serve as a model for a more general approach to enhance the strength and toughness of other biomaterials. PMID:19390040

Lee, Seung-Mo; Pippel, Eckhard; Gösele, Ulrich; Dresbach, Christian; Qin, Yong; Chandran, C Vinod; Bräuniger, Thomas; Hause, Gerd; Knez, Mato

2009-04-24

204

Gumfooted lines in black widow cobwebs and the mechanical properties of spider capture silk.  

PubMed

Orb-weaving spiders produce webs using two types of silk that have radically different mechanical properties. The dragline silk used to construct the supporting frame and radii of the web is stiff and as strong as steel, while the capture spiral is much weaker but more than ten times as extensible. This remarkable divergence in mechanical properties has been attributed to the aqueous glue that coats the capture spiral, which is thought to decrease capture spiral stiffness and increase its extensibility. However, discerning the effect of the aqueous glue on fiber performance is complicated because dragline silk and the capture spiral are assembled from different proteins, which may also affect mechanical performance. Here, we use the sticky gumfooted lines of black widow cobwebs to test the effect of the addition of aqueous glue on the mechanical properties of dragline silk. We also surveyed orb-webs spun by a broad range of species for bundles of looped silk. Such bundles, termed windlasses, have been thought to increase capture spiral extensibility by "paying out" additional lengths of silk. Our results suggest that neither plasticization of silk by aqueous glue nor excess silk in windlasses can by themselves account for the remarkable extensibility of orb-weaver capture silk compared to other spider silks. This argues that the unique amino acid motifs of the flagelliform fibroins that constitute the core of the capture spiral play an essential role in capture silk's extreme extensibility. PMID:16351953

Blackledge, Todd A; Summers, Adam P; Hayashi, Cheryl Y

2005-01-21

205

Silk fibroin microtubes for blood vessel engineering  

PubMed Central

Currently available synthetic grafts demonstrate moderate success at the macrovascular level, but fail at the microvascular scale (<6 mm inner diameter). We report on the development of silk fibroin microtubes for blood vessel repair with several advantages over existing scaffold materials/designs. These microtubes were prepared by dipping straight lengths of stainless steel wire into aqueous silk fibroin, where the addition of poly(ethylene oxide) (PEO) enabled control of microtube porosity. The microtube properties were characterized in terms of pore size, burst strength, protein permeability, enzymatic degradation, and cell migration. Low porosity microtubes demonstrated superior mechanical properties in terms of higher burst pressures, but displayed poor protein permeability; whereas higher porosity tubes had lower burst strengths but increased permeability and enhanced protein transport. The microtubes also exhibited cellular barrier functions as low porosity tubes prevented outward migration of GFP-transduced HUVECs, while the high porosity microtubes allowed a few cells per tube to migrate outward during perfusion. When combined with the biocompatible and suturability features of silk fibroin, these results suggest that silk microtubes, either implanted directly or preseeded with cells, are an attractive biomaterial for microvascular grafts.

Lovett, Michael L.; Cannizzaro, Christopher; Daheron, Laurence; Messmer, Brady; Vunjak-Novakovic, Gordana; Kaplan, David L.

2009-01-01

206

Biospinning by silkworms: silk fiber matrices for tissue engineering applications.  

PubMed

The mechanism of biospinning of natural silk fibers has been an open issue for decades. In this report a natural bio-polymeric matrix based on biospun silk fibers obtained from Antheraea mylitta, a wild non-mulberry tropical tasar silkworm, is put forward for potential applications. This report deals with the conformational transitions of silk fibroin during the biospinning process and its potential to support cell adherence and proliferation. The silk fibers obtained were aligned into linear, mixed or random patterns forming interconnected, macroporous three-dimensional matrices. The matrices were morphologically and functionally characterized with respect to fiber diameter, crystallinity, mechanical strength and biocompatibility using feline fibroblast cells. Drawn silk fibers showed enhanced stability to protease treatment in comparison with naturally occurring native gland fibroin protein. A viability assay suggested biocompatibility of these matrices in vitro. Fluorescence and confocal microscopy indicated normal cell attachment, spreading and proliferation on these biospun silk matrices. The results provided evidence for the use of biospun silk matrices as natural, inexpensive and alternative substrata for tissue engineering applications. PMID:19716447

Mandal, Biman B; Kundu, Subhas C

2009-08-27

207

Evidence for antimicrobial activity associated with common house spider silk  

PubMed Central

Background Spider silk is one of the most versatile materials in nature with great strength and flexibility. Native and synthetically produced silk has been used in a wide range of applications including the construction of artificial tendons and as substrates for human cell growth. In the literature there are anecdotal reports that suggest that native spider silk may also have antimicrobial properties. Findings In this study we compared the growth of a Gram positive and a Gram negative bacterium in the presence and absence of silk produced by the common house spider Tegenaria domestica. We demonstrate that native web silk of Tegenaria domestica can inhibit the growth of the Gram positive bacterium, Bacillus subtilis. No significant inhibition of growth was detected against the Gram negative bacterium, Escherichia coli. The antimicrobial effect against B. subtilis appears to be short lived thus the active agent potentially acts in a bacteriostatic rather than bactericidal manner. Treatment of the silk with Proteinase K appears to reduce the ability to inhibit bacterial growth. This is consistent with the active agent including a protein element that is denatured or cleaved by treatment. Tegenaria silk does not appear to inhibit the growth of mammalian cells in vitro thus there is the potential for therapeutic applications.

2012-01-01

208

Piriform Spider Silk Sequences Reveal Unique Repetitive Elements.  

PubMed

Orb-weaving spider silk fibers are assembled from very large, highly repetitive proteins. The repeated segments contain, in turn, short, simple, and repetitive amino acid motifs that account for the physical and mechanical properties of the assembled fiber. Of the six orb-weaver silk fibroins, the piriform silk that makes the attachment discs, which lashes the joints of the web and attaches dragline silk to surfaces, has not been previously characterized. Piriform silk protein cDNAs were isolated from phage libraries of three species: A. trifasciata , N. clavipes , and N. cruentata . The deduced amino acid sequences from these genes revealed two new repetitive motifs: an alternating proline motif, where every other amino acid is proline, and a glutamine-rich motif of 6-8 amino acids. Similar to other spider silk proteins, the repeated segments are large (>200 amino acids) and highly homogenized within a species. There is also substantial sequence similarity across the genes from the three species, with particular conservation of the repetitive motifs. Northern blot analysis revealed that the mRNA is larger than 11 kb and is expressed exclusively in the piriform glands of the spider. Phylogenetic analysis of the C-terminal regions of the new proteins with published spidroins robustly shows that the piriform sequences form an ortholog group. PMID:20954740

Perry, David J; Bittencourt, Daniela; Siltberg-Liberles, Jessica; Rech, Elibio L; Lewis, Randolph V

2010-10-18

209

Extraction conditions of Antheraea mylitta sericin with high yields and minimum molecular weight degradation.  

PubMed

Although the technique for extracting the Bombyx mori sericin has been extensively known, the extraction of sericin from wild-silkworm cocoons is not yet standardized. The aim of this study was to find the optimal conditions for the extraction of sericin from Antheraea mylitta cocoons, with high yields and minimum degradation. We attempted to apply various protocols for the extraction of the A. mylitta sericin (AmS). Among these, we found that the extraction of AmS with a sodium carbonate solution exhibited the highest yield except the conventional soap-alkali extraction. To find the optimal conditions for the AmS extraction with the sodium carbonate, we changed the concentration of sodium carbonate and the treatment time. With an increase in the sodium carbonate concentration and the extraction time, the yield of AmS increased, but the molecular weight (MW) of AmS decreased. Considering the yield, molecular weight distribution (MWD) and amino acid composition of AmS, we suggest that the optimal conditions for the AmS extraction require treatment with 0.02 M sodium carbonate and boiling for 60 min. PMID:23026092

Yun, Haesung; Oh, Hanjin; Kim, Moo Kon; Kwak, Hyo Won; Lee, Jeong Yun; Um, In Chul; Vootla, Shyam Kumar; Lee, Ki Hoon

2012-09-28

210

Evaluation of an injectable silk fibroin enhanced calcium phosphate cement loaded with human recombinant bone morphogenetic protein-2 in ovine lumbar interbody fusion.  

PubMed

The objective of this study was to investigate the efficacy of an injectable calcium phosphate cement/silk fibroin/human recombinant bone morphogenetic protein-2 composite (CPC/SF/rhBMP-2) in an ovine interbody fusion model. Twenty-four mature sheep underwent anterior lumbar interbody fusion at the levels of L1/2, L3/4, and L5/6 with random implantation of CPC/SF, CPC/rhBMP-2, CPC/SF/rhBMP-2, or autogenous iliac bone. After the sheep were sacrificed, the fusion segments were evaluated by manual palpation, CT scan, undestructive biomechanical testing, undecalcified histology, and histomorphology. The fusion rates of CPC/SF/rhBMP-2 were 55.56% and 77.78% at 6 and 12 months, respectively. The fusion was superior to all the biomaterial grafts in stiffness, and reached the same stiffness as the autograft at 12 months. The new bone formation was less than autograft at 6 months, but similar with that at 12 months. However, the ceramic residue volume of CPC/SF/rhBMP-2 was significantly decreased compared with CPC/SF and CPC/rhBMP-2 at both times. The results indicated that CPC/SF/rhBMP-2 composite had excellent osteoconduction and osteoinduction, and balanced degradation and osteogenesis. PMID:21381189

Gu, Yong; Chen, Liang; Yang, Hui-Lin; Luo, Zong-Ping; Tang, Tian-Si

2011-03-04

211

Evolution of supercontraction in spider silk: structure-function relationship from tarantulas to orb-weavers.  

PubMed

Spider silk is a promising biomaterial with impressive performance. However, some spider silks also 'supercontract' when exposed to water, shrinking by up to ?50% in length. Supercontraction may provide a critical mechanism to tailor silk properties, both for future synthetic silk production and by the spiders themselves. Several hypotheses are proposed for the mechanism and function of supercontraction, but they remain largely untested. In particular, supercontraction may result from a rearrangement of the GPGXX motif within the silk proteins, where G represents glycine, P proline and X is one of a small subset of amino acids. Supercontraction may prevent sagging in wet orb-webs or allow spiders to tailor silk properties for different ecological functions. Because both the molecular structures of silk proteins and how dragline is used in webs differ among species, we can test these hypotheses by comparing supercontraction of silk across diverse spider taxa. In this study we measured supercontraction in 28 spider taxa, ranging from tarantulas to orb-weaving spiders. We found that silk from all species supercontracted, except that of most tarantulas. This suggests that supercontraction evolved at least with the origin of the Araneomorphae, over 200 million years ago. We found differences in the pattern of evolution for two components of supercontraction. Stress generated during supercontraction of a restrained fiber is not associated with changes in silk structure and web architecture. By contrast, the shrink of unrestrained supercontracting fibers is higher for Orbiculariae spiders, whose silk contains high ratios of GPGXX motifs. These results support the hypothesis that supercontraction is caused by a rearrangement of GPGXX motifs in silk, and that it functions to tailor silk material properties. PMID:20889831

Boutry, Cecilia; Blackledge, Todd Alan

2010-10-15

212

Electrospun silk-BMP-2 scaffolds for bone tissue engineering  

Microsoft Academic Search

Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and\\/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk\\/PEO\\/BMP-2,

Chunmei Li; Charu Vepari; Hyoung-Joon Jin; Hyeon Joo Kim; David L. Kaplan

2006-01-01

213

Isolation of a Clone Encoding a Second Dragline Silk Fibroin. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account...

M. B. Hinnman R. V. Lewis

1992-01-01

214

Variation in the material properties of spider dragline silk across species  

Microsoft Academic Search

Spiders produce high performance fibers that compare favorably with the best manmade fibers in strength and toughness. The\\u000a amino acid sequences of silk proteins have been determined for a number of silk types and species, revealing extensive variation.\\u000a This variation in sequence is hypothesized to confer different material properties. However, the material properties of silk\\u000a have been characterized from only

B. O. Swanson; T. A. Blackledge; J. Beltrán; C. Y. Hayashi

2006-01-01

215

The Digital Silk Road  

Microsoft Academic Search

Existing and proposed mechanisms for digital money all require large overhead to transfer money between parties. This overhead makes them unsuitable for extremely low cost activities, such as delivering and routing packets. The digital silk road is a proposed money system with extremely low transaction cost built into the communication protocols. The money introduced by this system is much more

Norman Hardy; Eric Dean Tribble

1993-01-01

216

Materials: Surprising strength of silkworm silk  

NASA Astrophysics Data System (ADS)

Commercial silkworm silk is presumed to be much weaker and less extensible than spider dragline silk, which has been hailed as a 'super-fibre'. But we show here that the mechanical properties of silkworm silks can approach those of spider dragline silk when reeled under controlled conditions. We suggest that silkworms might be able to produce threads that compare well with spider silk by changing their spinning habits, rather than by having their silk genes altered.

Shao, Zhengzhong; Vollrath, Fritz

2002-08-01

217

Flexibility regeneration of silk fibroin in vitro  

PubMed Central

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were firstly prepared by slow drying process. Then the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared to the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nano-assembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.

Zhang, Cencen; Song, Dawei; Lu, Qiang; Hu, Xiao; Kaplan, David L; Zhu, Hesun

2012-01-01

218

Stabilization of Enzymes in Silk Films  

PubMed Central

Material systems are needed that promote stabilization of entrained molecules, such as enzymes or therapeutic proteins, without destroying their activity. We demonstrate that the unique structure of silk fibroin protein, when assembled into the solid state, establishes an environment that is conducive to the stabilization of entrained proteins. Enzymes (glucose oxidase, lipase and horseradish peroxidase) entrapped in these films over ten months retained significant activity, even when stored at 37°C, and in the case of glucose oxidase did not lose any activity. Further, the mode of processing of the silk protein into the films could be correlated to the stability of the enzymes. The relationship between processing and stability offers a large suite of conditions within which to optimize such stabilization processes. Overall, the techniques reported here result in materials that stabilize enzymes to a remarkable extent, without the need for cryoprotectants, emulsifiers, covalent immobilization or other treatments. Further, these systems are amenable to optical characterization, environmental distribution without refrigeration, are ingestible, and offer potential use in vivo, since silk materials are biocompatible and FDA approved, degradable with proteases and currently used in biomedical devices.

Lu, Shenzhou; Wang, Xiaoqin; Lv, Qiang; Hu, Xiao; Uppal, Neha; Omenetto, Fiorenzo

2009-01-01

219

Invited review the coiled coil silk of bees, ants, and hornets.  

PubMed

In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is ?-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks. PMID:21830200

Sutherland, Tara D; Weisman, Sarah; Walker, Andrew A; Mudie, Stephen T

2011-08-09

220

Characterisation of phenol oxidase and peroxidase from maize silk.  

PubMed

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non-browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o-diphenolic substrates, was abundant only in browning silk, and low or absent in non-browning silk. Pollination increased POD but not PPO activity. Isoelectric-focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN(3), EDTA, KCN, and L-cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination. PMID:20522176

Sukalovi?, V Hadzi-Taskovi?; Veljovi?-Jovanovi?, S; Maksimovi?, J Dragisi?; Maksimovi?, V; Paji?, Z

2010-05-01

221

Transdermal Delivery Devices: Fabrication, Mechanics and Drug Release from Silk.  

PubMed

Microneedles are a relatively simple, minimally invasive and painless approach to deliver drugs across the skin. However, there remain limitations with this approach because of the materials most commonly utilized for such systems. Silk protein, with tunable and biocompatibility properties, is a useful biomaterial to overcome the current limitations with microneedles. Silk devices preserve drug activity, offer superior mechanical properties and biocompatibility, can be tuned for biodegradability, and can be processed under aqueous, benign conditions. In the present work, the fabrication of dense microneedle arrays from silk with different drug release kinetics is reported. The mechanical properties of the microneedle patches are tuned by post-fabrication treatments or by loading the needles with silk microparticles, to increase capacity and mechanical strength. Drug release is further enhanced by the encapsulation of the drugs in the silk matrix and coating with a thin dissolvable drug layer. The microneedles are used on human cadaver skin and drugs are delivered successfully. The various attributes demonstrated suggest that silk-based microneedle devices can provide significant benefit as a platform material for transdermal drug delivery. PMID:23653252

Raja, Waseem K; Maccorkle, Scott; Diwan, Izzuddin M; Abdurrob, Abdurrahman; Lu, Jessica; Omenetto, Fiorenzo G; Kaplan, David L

2013-05-01

222

Relationships between supercontraction and mechanical properties of spider silk.  

PubMed

Typical spider dragline silk tends to outperform other natural fibres and most man-made filaments. However, even small changes in spinning conditions can have large effects on the mechanical properties of a silk fibre as well as on its water uptake. Absorbed water leads to significant shrinkage in an unrestrained dragline fibre and reversibly converts the material into a rubber. This process is known as supercontraction and may be a functional adaptation for the silk's role in the spider's web. Supercontraction is thought to be controlled by specific motifs in the silk proteins and to be induced by the entropy-driven recoiling of molecular chains. In analogy, in man-made fibres thermal shrinkage induces changes in mechanical properties attributable to the entropy-driven disorientation of 'unfrozen' molecular chains (as in polyethylene terephthalate) or the 'broken' intermolecular hydrogen bonds (as in nylons). Here we show for Nephila major-ampullate silk how in a biological fibre the spinning conditions affect the interplay between shrinkage and mechanical characteristics. This interaction reveals design principles linking the exceptional properties of silk to its molecular orientation. PMID:16299506

Liu, Yi; Shao, Zhengzhong; Vollrath, Fritz

2005-11-20

223

The effects of corn silk on glycaemic metabolism  

PubMed Central

Background Corn silk contains proteins, vitamins, carbohydrates, Ca, K, Mg and Na salts, fixed and volatile oils, steroids such as sitosterol and stigmasterol, alkaloids, saponins, tannins, and flavonoids. Base on folk remedies, corn silk has been used as an oral antidiabetic agent in China for decades. However, the hypoglycemic activity of it has not yet been understood in terms of modern pharmacological concepts. The purpose of this study is to investigate the effects of corn silk on glycaemic metabolism. Methods Alloxan and adrenalin induced hyperglycemic mice were used in the study. The effects of corn silk on blood glucose, glycohemoglobin (HbA1c), insulin secretion, damaged pancreatic ?-cells, hepatic glycogen and gluconeogenesis in hyperglycemic mice were studied respectively. Results After the mice were orally administered with corn silk extract, the blood glucose and the HbA1c were significantly decreased in alloxan-induced hyperglycemic mice (p < 0.05, p < 0.01, respectively), while the level of insulin secretionn was markedly elevated in alloxa-induced hyperglycemic mice (p < 0.05). The alloxan-damaged pancreatic ?-cells of the mice were partly recovered gradually after the mice were administered with corn silk extract 15 days later. Also, the body weight of the alloxan-induced hyperglycemic mice was increased gradually. However, ascension of blood glucose induced by adrenalin and gluconeogenesis induced by L-alanine were not inhibited by corn silk extract treatment (p > 0.05). Although corn silk extract increased the level of hepatic glycogen in the alloxan-induced hyperglycemic mice, there was no significant difference between them and that of the control group(p > 0.05). Conclusion Corn silk extract markedly reduced hyperglycemia in alloxan-induced diabetic mice. The action of corn silk extract on glycaemic metabolism is not via increasing glycogen and inhibiting gluconeogenesis but through increasing insulin level as well as recovering the injured ?-cells. The results suggest that corn silk extract may be used as a hypoglycemic food or medicine for hyperglycemic people in terms of this modern pharmacological study.

2009-01-01

224

Properties and potential medical applications of silk fibers produced by Rothischildia lebeau.  

PubMed

Rothischildia lebeau which belongs to the Saturniidae family of silk-producing insects secretes protein fibers with properties between that of the Bombyx mori and the common wild silks. Traditionally, wild silks produced by insects such as Antheraea mylitta are considerably coarser and have inferior tensile properties than the domesticated and most commonly used silk produced by B. mori. Recently, it has been demonstrated that some of the wild silks have unique properties and preferable for medical applications. Wild silks are comparatively easier to rear, produce larger cocoons, and could have unique properties. In this research, the structure and properties of the silk fibers produced by R. lebeau were studied and the potential of using the fibers for medical applications was investigated. Fibers produced by R. lebeau had average tensile strength of 3.3?g/den, similar to that of wild silks but lower than that of the B. mori silk. R. lebeau fibers were biocompatible and showed potential to be useful for tissue engineering and other medical applications. PMID:23594071

Reddy, Narendra; Jiang, Qiuran; Yang, Yiqi

2012-08-28

225

Tuning chemical and physical cross-links in silk electrogels for morphological analysis and mechanical reinforcement.  

PubMed

Electrochemically controlled, reversible assembly of biopolymers into hydrogel structures is a promising technique for on-demand cell or drug encapsulation and release systems. An electrochemically sol-gel transition has been demonstrated in regenerated Bombyx mori silk fibroin, offering a controllable way to generate biocompatible and reversible adhesives and other biomedical materials. Despite the involvement of an electrochemically triggered electrophoretic migration of the silk molecules, the mechanism of the reversible electrogelation remains unclear. It is, however, known that the freshly prepared silk electrogels (e-gels) adopt a predominantly random coil conformation, indicating a lack of cross-linking as well as thermal, mechanical, and morphological stabilities. In the present work, the tuning of covalent and physical ?-sheet cross-links in silk hydrogels was studied for programming the structural properties. Scanning electron microscopy (SEM) revealed delicate morphology, including locally aligned fibrillar structures, in silk e-gels, preserved by combining glutaraldehyde-cross-linking and ethanol dehydration. Fourier transform infrared (FTIR) spectroscopic analysis of either electrogelled, vortex-induced or spontaneously formed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non-?-sheet-dominated and ?-sheet-dominated states. Dynamic oscillatory rheology confirmed the mechanical reinforcement of silk e-gels provided by controlled chemical and physical cross-links. The selective incorporation of either chemical or physical or both cross-links into the electrochemically responsive, originally unstructured silk e-gel should help in the design for electrochemically responsive protein polymers. PMID:23859710

Lin, Yinan; Xia, Xiaoxia; Shang, Ke; Elia, Roberto; Huang, Wenwen; Cebe, Peggy; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L

2013-07-16

226

Silk constructs for delivery of muskuloskeletal therapeutics  

PubMed Central

Silk fibroin (SF) is a biopolymer with distinguishing features from many other bio- as well as synthetic polymers. From a biomechanical and drug delivery perspective, SF combines remarkable versatility for scaffolding (solid implants, hydrogels, threads, solutions), with advanced mechanical properties and good stabilization and controlled delivery of entrapped protein and small molecule drugs, respectively. It is this combination of mechanical and pharmaceutical features which render SF so exciting for biomedical applications. his pattern along with the versatility of this biopolymer have been translated into progress for musculoskeletal applications. We review the use and potential of silk fibroin for systemic and localized delivery of therapeutics in diseases affecting the musculoskeletal system. We also present future directions for this biopolymer as well as the necessary research and development steps for their achievement.

Meinel, Lorenz; Kaplan, David L.

2012-01-01

227

Materials: Surprising strength of silkworm silk  

Microsoft Academic Search

Commercial silkworm silk is presumed to be much weaker and less extensible than spider dragline silk, which has been hailed as a 'super-fibre'. But we show here that the mechanical properties of silkworm silks can approach those of spider dragline silk when reeled under controlled conditions. We suggest that silkworms might be able to produce threads that compare well with

Zhengzhong Shao; Fritz Vollrath

2002-01-01

228

Nanoconfinement of spider silk fibrils begets superior strength, extensibility, and toughness.  

PubMed

Silk is an exceptionally strong, extensible, and tough material made from simple protein building blocks. The molecular structure of dragline spider silk repeat units consists of semiamorphous and nanocrystalline ?-sheet protein domains. Here we show by a series of computational experiments how the nanoscale properties of silk repeat units are scaled up to create macroscopic silk fibers with outstanding mechanical properties despite the presence of cavities, tears, and cracks. We demonstrate that the geometric confinement of silk fibrils to diameters of 50 ± 30 nm is critical to facilitate a powerful mechanism by which hundreds of thousands of protein domains synergistically resist deformation and failure to provide enhanced strength, extensibility, and toughness at the macroscale, closely matching experimentally measured mechanical properties. Through this mechanism silk fibers exploit the full potential of the nanoscale building blocks, regardless of the details of microscopic loading conditions and despite the presence of large defects. Experimental results confirm that silk fibers are composed of silk fibril bundles with diameters in the range of 20-150 nm, in agreement with our predicted length scale. Our study reveals a general mechanism to map nanoscale properties to the macroscale and provides a potent design strategy toward novel fiber and bulk nanomaterials through hierarchical structures. PMID:21967633

Giesa, Tristan; Arslan, Melis; Pugno, Nicola M; Buehler, Markus J

2011-10-21

229

Non-bioengineered silk gland fibroin micromolded matrices to study cell-surface interactions.  

PubMed

Micropatterning/micromolding of protein molecules has played a significant role in developing biosensors, micro arrays, and tissue engineering devices for cellular investigations. Relevantly, there have been ample scopes for silk to be used as natural biomaterial in tissue engineering applications due to its attractive properties such as slow-controllable degradation, mechanical robustness, and inherent biocompatibility. In this paper, we report the fabrication of micromolded silk fibroin matrices, which have essentially been utilized to study cell-surface interactions. Fibroin protein has been isolated from the silk glands of nonmulberry Indian tropical tasar silkworms, Antheraea mylitta. The surface uniformity has been investigated using atomic force microscopy following the fabrication of silk micromolds. Subsequently, cellular interactions in terms of cell attachment, spreading, mitochondrial activity and proliferation have been studied in vitro using feline fibroblasts. Results have indicated a long term stability of patterns in micromolded silk matrices and negligible swelling. The versatility of described silk dissolution method coupled with ability to process large amount of silk protein into micromolded matrices and controllable surface topology may augment the desirability of silk fibroin as a natural biomaterial for bioengineering and biotechnological applications. PMID:19058012

Mandal, Biman B; Das, Tamal; Kundu, S C

2009-04-01

230

Analysis of major ampullate silk cDNAs from two non-orb-weaving spiders.  

PubMed

Compared to other arthropods, spiders are unique in their use of silk throughout their life span and the extraordinary mechanical properties of the silk threads they produce. Studies on orb-weaving spider silk proteins have shown that silk proteins are composed of highly repetitive regions, characterized by alanine and glycine-rich units. We have isolated and sequenced four partial cDNA clones representing major ampullate spider silk gene transcripts from two non-orb weavers: three for Kukulcania hibernalis and one for Agelenopsis aperta. These cDNA sequences were compared to each other, as well as to the previously published orb-weaver silk gene sequences. The results indicate that the repeats encoding conserved amino acid motifs such as polyA and polyGA that are characteristic of some orb-weaving spider silks are also found in some of the cDNAs reported in this study. However, we also found other motifs such as polyGS and polyGV in the cDNA sequences from the two non-orb-weaving spiders. The amino acid composition of the silk gland extracts shows that alanine and glycine are the major components of the silk of these two non-orb weavers as is the case in orb-weaver silks. Sequence alignment shows that A. aperta's cDNA displays a C-terminal encoding region that is about 44% similar to the one present in N. clavipes's MaSp1 cDNA. In addition, as previously observed for spider silk sequences, the analysis of the codon usage for these four cDNAs demonstrates a bias for A or T in the wobble base position. PMID:15132643

Tian, Maozhen; Liu, Congzhou; Lewis, Randolph

231

Synthetic spider silk production on a laboratory scale.  

PubMed

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks. PMID:22847722

Hsia, Yang; Gnesa, Eric; Pacheco, Ryan; Kohler, Kristin; Jeffery, Felicia; Vierra, Craig

2012-07-18

232

Shear-induced rigidity in spider silk glands  

NASA Astrophysics Data System (ADS)

We measure the elastic stiffnesses of the concentrated viscous protein solution of the dehydrated Nephila clavipes major ampullate gland with Brillouin light scattering. The glandular material shows no rigidity but possesses a tensile stiffness similar to that of spider silk. We show, however, that with application of a simple static shear, the mechanical properties of the spider gland protein mixture can be altered irreversibly, lowering symmetry and enabling shear waves to be supported, thus, giving rise to rigidity and yielding elastic properties similar to those of the naturally spun (i.e., dynamically sheared) silk.

Koski, Kristie J.; McKiernan, Keri; Akhenblit, Paul; Yarger, Jeffery L.

2012-09-01

233

Changes in growth and cell wall extensibility of maize silks following pollination  

PubMed Central

In response to pollination maize silks undergo an accelerated process of senescence which involves an inhibition of elongation. To gain insight into the mechanism underlying this growth response, the relationships among silk elongation kinetics, cell wall biophysical properties, pollen tube growth, and expansin protein abundance were investigated. The inhibition of silk elongation became apparent beyond 12?h after pollination. Pollinated walls were less responsive in assays of extension induced by pollen ?-expansin. Expansin protein abundance and endogenous expansin activity were not considerably reduced after pollination. Silk wall plastic compliance was significantly reduced 6?h post-pollination and beyond, suggesting that the wall undergoes structural modifications leading to its rigidification in response to pollination. The reduction in the plastic compliance occurred locally and progressively, shortly after pollen tubes traversed through a region of silk. Though numerous pollen grains germinated and initiated pollen tubes at the silk tip, the density of pollen tubes gradually declined along the length of the silk and only 1–2 reached the ovary even 24?h after pollination. These results support the notion that pollination-induced cell wall rigidification plays multiple roles in maize reproduction, including inhibition of silk growth and prevention of polyspermy.

Sella Kapu, Nuwan U.; Cosgrove, Daniel J.

2010-01-01

234

Changes in growth and cell wall extensibility of maize silks following pollination.  

PubMed

In response to pollination maize silks undergo an accelerated process of senescence which involves an inhibition of elongation. To gain insight into the mechanism underlying this growth response, the relationships among silk elongation kinetics, cell wall biophysical properties, pollen tube growth, and expansin protein abundance were investigated. The inhibition of silk elongation became apparent beyond 12 h after pollination. Pollinated walls were less responsive in assays of extension induced by pollen beta-expansin. Expansin protein abundance and endogenous expansin activity were not considerably reduced after pollination. Silk wall plastic compliance was significantly reduced 6 h post-pollination and beyond, suggesting that the wall undergoes structural modifications leading to its rigidification in response to pollination. The reduction in the plastic compliance occurred locally and progressively, shortly after pollen tubes traversed through a region of silk. Though numerous pollen grains germinated and initiated pollen tubes at the silk tip, the density of pollen tubes gradually declined along the length of the silk and only 1-2 reached the ovary even 24 h after pollination. These results support the notion that pollination-induced cell wall rigidification plays multiple roles in maize reproduction, including inhibition of silk growth and prevention of polyspermy. PMID:20656797

Kapu, Nuwan U Sella; Cosgrove, Daniel J

2010-07-23

235

Accelerated In Vitro Degradation of Optically Clear Low ?-Sheet Silk Films by Enzyme-Mediated Pretreatment  

PubMed Central

Purpose: To design patterned, transparent silk films with fast degradation rates for the purpose of tissue engineering corneal stroma. Methods: ?-sheet (crystalline) content of silk films was decreased significantly by using a short water annealing time. Additionally, a protocol combining short water annealing time with enzymatic pretreatment of silk films with protease XIV was developed. Results: Low ?-sheet content (17%–18%) and enzymatic pretreatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced ?-sheet content and enzymatic pretreatment, and overall degradation rate of the protein films. Conclusions: The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. Translational Relevance: Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment, and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration.

Shang, Ke; Rnjak-Kovacina, Jelena; Lin, Yinan; Hayden, Rebecca S.; Tao, Hu; Kaplan, David L.

2013-01-01

236

NMR Studies of Molecular Orientation and Dynamics in Spider silk  

NASA Astrophysics Data System (ADS)

Spider dragline silk has a unique combination of strength and extensibility that has been difficult to achieve in synthetic polymer fibres and has inspired industrial efforts to produce genetically engineered analogues. In light of these efforts elsewhere, we describe solid-state NMR experiments that elucidate the molecular structure and dynamics of this remarkable material. These experiments include the use of a 2-D exchange NMR experiment known as DECODER in which the sample is reoriented through a discrete angle during the mixing time. This experiment allows a reconstruction of the orientation distribution of the protein backbone. Our data is well described by a two-component distribution where the protein backbones of both components are preferentially aligned along the silk fibre. This experiment is also sensitive to molecular motion on a wide range of time-scales, and is employed to study changes in the silk as a function of fibre extension and hydration. Hydrated silk undergoes a remarkable phenomena known as supercontraction where fibres shrink by up to 50% in length while swelling in diameter. DECODER NMR of fully and partially supercontracted silk reveals that supercontraction occurs through a process of local phase transitions where water disrupts inter- and intra-chain hydrogen bonds.

Michal, Carl; Eles, Philip

2004-05-01

237

Uncovering spider silk nanocrystalline variations that facilitate wind-induced mechanical property changes.  

PubMed

Spider major ampullate (MA) silk varies in mechanical properties when spun in different environments. Amino acid compositional changes induced by variations in MaSp1 and MaSp2 expression, and various biochemical and physiological glandular processes induce silk property variability. Quantifying the contributions of these mechanisms on silk variability may facilitate the development of silk biomimetics. Wind is a medium that induces variations in MA silk mechanics. We exposed the spider Cyclosa mulmeinensis to wind and measured the amino acid composition, tensile mechanics, and crystalline structure of its MA silk using HPLC, tensile tests, and X-ray diffraction. We found the mechanical properties of MA silks from spiders exposed to wind to differ from unexposed spiders. The amino acid compositions did not differ, but X-ray diffraction found a lower crystal density and greater ?-sheet alignment relative to the fiber axis in the silks of spiders exposed to wind. We found no evidence that the mechanical property variations were a product of profound changes to the alignment of the protein within the amorphous region. We conclude that variations in the density and alignment of the crystalline ?-sheets, probably accompanied by some alignment change in the amorphous region as a result of "stretching" during spinning of the silk, probably explains the mechanical property variations that we found across treatment subgroups. As C. mulmeinensis MA silk increases both in strength and elasticity when the spiders are exposed to wind, bioengineers may consider it as a model for the development of high-performance silk biomimetics. PMID:23947397

Blamires, Sean J; Wu, Chao-Chia; Wu, Chung-Lin; Sheu, Hwo-Shuenn; Tso, I-Min

2013-09-05

238

Increasing silk fibre strength through heterogeneity of bundled fibrils.  

PubMed

Can naturally arising disorder in biological materials be beneficial? Materials scientists are continuously attempting to replicate the exemplary performance of materials such as spider silk, with detailed techniques and assembly procedures. At the same time, a spider does not precisely machine silk-imaging indicates that its fibrils are heterogeneous and irregular in cross section. While past investigations either focused on the building material (e.g. the molecular scale protein sequence and behaviour) or on the ultimate structural component (e.g. silk threads and spider webs), the bundled structure of fibrils that compose spider threads has been frequently overlooked. Herein, I exploit a molecular dynamics-based coarse-grain model to construct a fully three-dimensional fibril bundle, with a length on the order of micrometres. I probe the mechanical behaviour of bundled silk fibrils with variable density of heterogenic protrusions or globules, ranging from ideally homogeneous to a saturated distribution. Subject to stretching, the model indicates that cooperativity is enhanced by contact through low-force deformation and shear 'locking' between globules, increasing shear stress transfer by up to 200 per cent. In effect, introduction of a random and disordered structure can serve to improve mechanical performance. Moreover, addition of globules allows a tuning of free volume, and thus the wettability of silk (with implications for supercontraction). These findings support the ability of silk to maintain near-molecular-level strength at the scale of silk threads, and the mechanism could be easily adopted as a strategy for synthetic fibres. PMID:23486175

Cranford, Steven W

2013-03-13

239

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk.  

PubMed

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract. PMID:24162473

Perea, G B; Riekel, C; Guinea, G V; Madurga, R; Daza, R; Burghammer, M; Hayashi, C; Elices, M; Plaza, G R; Pérez-Rigueiro, J

2013-10-28

240

Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk  

PubMed Central

Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the –GGX– and –GPG– motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.

Perea, G. B.; Riekel, C.; Guinea, G. V.; Madurga, R.; Daza, R.; Burghammer, M.; Hayashi, C.; Elices, M.; Plaza, G. R.; Perez-Rigueiro, J.

2013-01-01

241

Physical characterization of functionalized spider silk: electronic and sensing properties  

NASA Astrophysics Data System (ADS)

This work explores functional, fundamental and applied aspects of naturally harvested spider silk fibers. Natural silk is a protein polymer where different amino acids control the physical properties of fibroin bundles, producing, for example, combinations of ?-sheet (crystalline) and amorphous (helical) structural regions. This complexity presents opportunities for functional modification to obtain new types of material properties. Electrical conductivity is the starting point of this investigation, where the insulating nature of neat silk under ambient conditions is described first. Modification of the conductivity by humidity, exposure to polar solvents, iodine doping, pyrolization and deposition of a thin metallic film are explored next. The conductivity increases exponentially with relative humidity and/or solvent, whereas only an incremental increase occurs after iodine doping. In contrast, iodine doping, optimal at 70?°C, has a strong effect on the morphology of silk bundles (increasing their size), on the process of pyrolization (suppressing mass loss rates) and on the resulting carbonized fiber structure (that becomes more robust against bending and strain). The effects of iodine doping and other functional parameters (vacuum and thin film coating) motivated an investigation with magic angle spinning nuclear magnetic resonance (MAS-NMR) to monitor doping-induced changes in the amino acid-protein backbone signature. MAS-NMR revealed a moderate effect of iodine on the helical and ?-sheet structures, and a lesser effect of gold sputtering. The effects of iodine doping were further probed by Fourier transform infrared (FTIR) spectroscopy, revealing a partial transformation of ?-sheet-to-amorphous constituency. A model is proposed, based on the findings from the MAS-NMR and FTIR, which involves iodine-induced changes in the silk fibroin bundle environment that can account for the altered physical properties. Finally, proof-of-concept applications of functionalized spider silk are presented for thermoelectric (Seebeck) effects and incandescence in iodine-doped pyrolized silk fibers, and metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers. In the latter case, we demonstrate the application of gold-sputtered neat spider silk to make four-terminal, flexible, ohmic contacts to organic superconductor samples.

Steven, Eden; Park, Jin Gyu; Paravastu, Anant; Branco Lopes, Elsa; Brooks, James S.; Englander, Ongi; Siegrist, Theo; Kaner, Papatya; Alamo, Rufina G.

2011-10-01

242

Physical Characterization of Functionalized Silk Material for Electronic Application and Devices  

NASA Astrophysics Data System (ADS)

Naturally harvested spider silk fibers are investigated for their physical properties under ambient, humidified, iodine-doped, pyrolized, sputtered gold and carbon nanotube coated conditions. The functional properties include: humidity activated conductivity; enhanced flexibility and carbon yield of pyrolized iodized silk fibers; full metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers; and high strain sensitivity of carbon nanotube coated silk fibers. Magic angle spinning nuclear magnetic resonance (MAS-NMR) and Fourier transform infrared spectroscopy (FTIR) are used to explore the nature of ambient and functionalized spider silk fiber, and significant changes in amino acid-protein backbone signature are correlated with gold sputtering, and iodine-doped conditions. The application of gold-sputtered neat spider silk fibers for making four terminal flexible, clean, ohmic contacts to organic superconductor samples and carbon nanotube coated silk fibers for heart pulse monitoring sensor are demonstrated. The role of silk thin film in organic thin film transistor will be briefly discussed.

Steven, Eden; Jobiliong, Eric; Park, Jin Gyu; Paravastu, Anant; Davidson, Michael; Baird, Michelle; Alamo, Rufina; Kaner, Papatya; Brooks, James; Siegrist, Theo

2012-02-01

243

Smart assembly of polymer fibers: lessons from major ampullate spider silk  

NASA Astrophysics Data System (ADS)

Studies of major ampullate silk (MAS), especially the secretions and fibers produced by the spider Nephila clavipes (golden orb weaver), have yielded several results of potential value to the materials scientist/engineer. There are lessons to be learned about synthesis, processing and microstructural design of high-tensile polymer fibers. The 'smart' aspect of silk production in nature concerns the ability of the spider to rapidly process a concentrated, viscous aqueous solution of silk protein (stored in the gland) into water-insoluble fiber on demand. This process centers on the assembly of a shear-sensitive supramolecular liquid crystalline phase by aggregation of the solubilized globular protein molecules.

Viney, Christopher

1996-02-01

244

Carbon nanotubes on a spider silk scaffold  

NASA Astrophysics Data System (ADS)

Understanding the compatibility between spider silk and conducting materials is essential to advance the use of spider silk in electronic applications. Spider silk is tough, but becomes soft when exposed to water. Here we report a strong affinity of amine-functionalised multi-walled carbon nanotubes for spider silk, with coating assisted by a water and mechanical shear method. The nanotubes adhere uniformly and bond to the silk fibre surface to produce tough, custom-shaped, flexible and electrically conducting fibres after drying and contraction. The conductivity of coated silk fibres is reversibly sensitive to strain and humidity, leading to proof-of-concept sensor and actuator demonstrations.

Steven, Eden; Saleh, Wasan R.; Lebedev, Victor; Acquah, Steve F. A.; Laukhin, Vladimir; Alamo, Rufina G.; Brooks, James S.

2013-09-01

245

Carbon nanotubes on a spider silk scaffold  

PubMed Central

Understanding the compatibility between spider silk and conducting materials is essential to advance the use of spider silk in electronic applications. Spider silk is tough, but becomes soft when exposed to water. Here we report a strong affinity of amine-functionalised multi-walled carbon nanotubes for spider silk, with coating assisted by a water and mechanical shear method. The nanotubes adhere uniformly and bond to the silk fibre surface to produce tough, custom-shaped, flexible and electrically conducting fibres after drying and contraction. The conductivity of coated silk fibres is reversibly sensitive to strain and humidity, leading to proof-of-concept sensor and actuator demonstrations.

Steven, Eden; Saleh, Wasan R.; Lebedev, Victor; Acquah, Steve F. A.; Laukhin, Vladimir; Alamo, Rufina G.; Brooks, James S.

2013-01-01

246

Silk materials--a road to sustainable high technology.  

PubMed

This review addresses the use of silk protein as a sustainable material in optics and photonics, electronics and optoelectronic applications. These options represent additional developments for this technology platform that compound the broad utility and impact of this material for medical needs that have been recently described in the literature. The favorable properties of the material certainly make a favorable case for the use of silk, yet serve as a broad inspiration to further develop biological foundries for both the synthesis and processing of Nature's materials for technological applications. PMID:22553118

Tao, Hu; Kaplan, David L; Omenetto, Fiorenzo G

2012-05-02

247

Total X-Ray Scattering of Spider Dragline Silk  

NASA Astrophysics Data System (ADS)

Total x-ray scattering measurements of spider dragline silk fibers from Nephila clavipes, Argiope aurantia, and Latrodectus hesperus all yield similar structure factors, with only small variations between the different species. Wide-angle x-ray scattering from fibers orientated perpendicular to the beam shows a high degree of anisotropy, and differential pair distribution functions obtained by integrating over wedges of the equatorial and meridian planes indicate that, on average, the majority (95%) of the atom-atom correlations do not extend beyond 1 nm. Futhermore, the atom-atom correlations between 1 and 3 nm are not associated with the most intense diffraction peaks at Q=1-2Å-1. Disordered molecular orientations along the fiber axis are consistent with proteins in similar structural arrangements to those in the equatorial plane, which may be associated with the silk’s greater flexibility in this direction.

Benmore, C. J.; Izdebski, T.; Yarger, J. L.

2012-04-01

248

Silk Polymer Designs for Improved Expression and Processing.  

National Technical Information Service (NTIS)

Silk fibers formed by insects and spiders are noted for their remarkable mechanical properties as well as their durability and biocompatibility. The exceptional solubility in vivo (20-30% w/v) of these proteins is dictated by both the need to produce soli...

D. L. Kaplan

2007-01-01

249

Silk matrix for tissue engineered anterior cruciate ligaments  

Microsoft Academic Search

A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy,

Gregory H Altman; Rebecca L Horan; Helen H Lu; Jodie Moreau; Ivan Martin; John C Richmond; David L Kaplan

2002-01-01

250

Silk matrix for tissue engineered anterior cruciate ligaments  

Microsoft Academic Search

A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy,

Gregory H. Altman; Rebecca L. Horana; Helen H. Lu; Jodie Moreau; John C. Richmond; David L. Kaplan

251

Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery  

PubMed Central

Background Degummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity. Methods In this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin. Results Gel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (?-form, type II ?-turn), while the other treatments produced more silk II (?-form, anti-parallel ?-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments. Conclusions Collectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.

2012-01-01

252

Spider Silk Spun and Integrated into Composites.  

National Technical Information Service (NTIS)

We gained significant new insights into the role of hydration for silks with our data allowing us to model in considerable detail the interaction between silk molecules and water. Our fibre and feedstock experiments demonstrated that the hierachical morph...

F. Vollrath

2009-01-01

253

Does the Giant Wood Spider Nephila pilipes Respond to Prey Variation by Altering Web or Silk Properties?  

Microsoft Academic Search

Recent studies demonstrated that orb-weaving spiders may alter web ar- chitectures, the amount of silk in webs, or the protein composition of silks in response to variation in amount or type of prey. In this study, we conducted food manipulations to examine three mechanisms by which orb-weaving spiders may adjust the performance of webs to vari- ation in prey by

I-Min Tso; Shu-Ya Chiang; Todd A. Blackledge

2007-01-01

254

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration  

Microsoft Academic Search

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae

Haq Abdul Shaik; Frantisek Sehnal

2009-01-01

255

Mechanical Response of Silk Crystalline Units from Force-Distribution Analysis  

PubMed Central

The outstanding mechanical toughness of silk fibers is thought to be caused by embedded crystalline units acting as cross links of silk proteins in the fiber. Here, we examine the robustness of these highly ordered ?-sheet structures by molecular dynamics simulations and finite element analysis. Structural parameters and stress-strain relationships of four different models, from spider and Bombyx mori silk peptides, in antiparallel and parallel arrangement, were determined and found to be in good agreement with x-ray diffraction data. Rupture forces exceed those of any previously examined globular protein many times over, with spider silk (poly-alanine) slightly outperforming Bombyx mori silk ((Gly-Ala)n). All-atom force distribution analysis reveals both intrasheet hydrogen-bonding and intersheet side-chain interactions to contribute to stability to similar extent. In combination with finite element analysis of simplified ?-sheet skeletons, we could ascribe the distinct force distribution pattern of the antiparallel and parallel silk crystalline units to the difference in hydrogen-bond geometry, featuring an in-line or zigzag arrangement, respectively. Hydrogen-bond strength was higher in antiparallel models, and ultimately resulted in higher stiffness of the crystal, compensating the effect of the mechanically disadvantageous in-line hydrogen-bond geometry. Atomistic and coarse-grained force distribution patterns can thus explain differences in mechanical response of silk crystals, opening up the road to predict full fiber mechanics.

Xiao, Senbo; Stacklies, Wolfram; Cetinkaya, Murat; Markert, Bernd; Grater, Frauke

2009-01-01

256

Silk-Based Gene Carriers with Cell Membrane-Destabilizing Peptides  

PubMed Central

Complexes of recombinant silk-polylysine molecules with ppTG1 peptide, a lysine-rich cell membrane-destabilizing peptide to bind plasmid DNA (pDNA), are designed as less-cytotoxic and highly efficient gene carriers. The peptide destabilizes the cell membrane and promotes gene transfer. Our particular interest is in how ppTG1 enhances transfection efficiency of the silk-based delivery system into human cells. Genetically engineered silk proteins containing polylysine and the monomeric and dimeric ppTG1 sequences are synthesized in Escherichia coli, followed by transfection experiments. The pDNA complexes of Silk-polylysine-ppTG1 dimer recombinant proteins prepared at an N/P 2 (the ratio of number of amines/ phosphates from pDNA) shows the highest transfection efficiency into human embryonic kidney (HEK) cells, the level of which is comparable to the transfection reagent Lipofectamine 2000. The assemblies show a globular morphology with an average hydrodynamic diameter of 99 nm and almost no beta-sheet structure. Additionally, the silk-based pDNA complexes demonstrate excellent DNase resistance as well as efficient release of the pDNA by enzymes that degrade silk proteins. Also, comparison with beta-sheet induced silk-based pDNA complexes indicates that the beta-sheet structure content of the silk sequence of the pDNA complexes controls the enzymatic degradation rate of the complexes, and hence can regulate the release profile of genes from the complexes. The bioengineered silk-based gene delivery vehicles containing cell membrane-destabilizing peptides are therefore concluded to have potential for a less-toxic and controlled-release gene delivery system.

Numata, Keiji; Kaplan, David L

2010-01-01

257

Discrimination of cultivated silk and wild silk by conventional instrumental analyses.  

PubMed

In Japan, recent trends have seen wild silk preferred over cultivated silk because of its texture. Some cases of fraud have occurred where cultivated silk garments are sold as wild silk. Samples from these cases, morphological observation using light microscope and polarized microscope have been conducted in forensic science laboratories. Sometimes scanning electron microscopy was also carried out. However, the morphology of silk shows quite wide variation, which makes it difficult to discriminate wild and cultivated silks by this method. In this report, silk discrimination was investigated using conventional instrumental analyses commonly available in forensic laboratories, such as Fourier-transfer infrared spectrometry (FT-IR), pyrolysis-gas chromatography/mass spectrometry (pyr-GC/MS) and differential thermal analysis (DTA). By FT-IR, cultivated and wild silk gave similar infrared spectra, but wild silk had a characteristic peak at 965 cm(-1) from the deformation vibration of the carbon-carbon double bond of the indole ring. Comparison of the pyrograms of cultivated and wild silk showed that wild silk had large indole and skatole peaks that cultivated silk did not, and these peaks might arise from tryptophan. The results of thermogravimetry/DTA showed that the endothermic peak was about 40 °C higher for wild silk than for cultivated silk. Using a combination of these results, cultivated and wild silk could be discriminated by common forensic instrumental techniques. PMID:23742990

Matsuyama, Yuji; Nagatani, Yoshiaki; Goto, Toshiyuki; Suzuki, Shinichi

2013-06-04

258

SILK: Scout Paths in the Linux Kernel  

Microsoft Academic Search

SILK stands for Scout In the Linux Kernel, and is a port of the Scout operating system to run as a Linux kernel mod- ule. SILK forms a replacement networking subsystem for standard Linux 2.4 kernels. Linux applications create and use Scout paths via the Linux socket interface with virtually no modifications to the applications themselves. SILK pro- vides Linux

Andy Bavier; Thiemo Voigt; Mike Wawrzoniak; Larry Peterson; Per Gunningberg

2001-01-01

259

Spectral analysis of induced color change on periodically nanopatterned silk films.  

PubMed

We demonstrate controllable structural color based on periodic nanopatterned 2D lattices in pure protein films of silk fibroin. We show here periodic lattices in silk fibroin films with feature sizes of hundreds of nanometers that exhibit different colors as a function of varying lattice spacing. Further, when varying the index of refraction contrast between the nanopatterned lattice and its surrounding environment by applying liquids on top of the lattices, colorimetric shifts are observed. The effect is characterized experimentally and theoretically and a simple example of glucose concentration sensing is presented. This is the first example of a functional sensor based on silk fibroin optics. PMID:19997366

Amsden, Jason J; Perry, Hannah; Boriskina, Svetlana V; Gopinath, Ashwin; Kaplan, David L; Dal Negro, Luca; Omenetto, Fiorenzo G

2009-11-01

260

Nephila clavipes Flagelliform silk-like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers.  

PubMed

Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength, though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength, with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers. PMID:23646825

Adrianos, Sherry L; Teulé, Florence; Hinman, Michael B; Jones, Justin A; Weber, Warner S; Yarger, Jeffery L; Lewis, Randolph V

2013-05-22

261

Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing  

PubMed Central

We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4°C (alpha-helix dominated silk I structure), to highest content of ~60% crystallinity at 100°C (beta-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature controlled regulation of water vapor, to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods which use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties.

Hu, Xiao; Shmelev, Karen; Sun, Lin; Gil, Eun-Seok; Park, Sang-Hyug; Cebe, Peggy; Kaplan, David L.

2011-01-01

262

Silk fibroin regulated mineralization of hydroxyapatite nanocrystals  

NASA Astrophysics Data System (ADS)

In the present study, silk fibroin was employed to regulate the mineralization of hydroxyapatite (HA) nanocrystals. The calcium phosphate crystals precipitated in the aqueous solution of silk fibroin at pH 8 and room temperature. The depositions collected at different reaction time were detected by X-ray diffraction analysis to investigate the mineralization process of calcium phosphate. The results indicated that fibroin protein could significantly promote the crystal growth of HA. The formed HA crystals were also studied by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The FTIR results revealed that the HA crystals are carbonate-substituted HA and compounded with fibroin. There are strong chemical interactions between HA and fibroin protein, which can be derived from the blue shift of amide II peak (from the position of 1517 1539 cm-1). TEM images showed that the mineralized nanofibrils in the composites are rod like in shape with the diameter of about 2 3 nm. Selected area electron diffraction patterns from the composites exhibit polycrystalline rings, which were well indexed as the HA phase with 0 0 2 preferential orientations.

Kong, X. D.; Cui, F. Z.; Wang, X. M.; Zhang, M.; Zhang, W.

2004-09-01

263

Structure and Properties of Nephila Clavipes Dragline Silk Polymer  

NASA Astrophysics Data System (ADS)

Silk, spun from an aqueous state at room temperature by a variety of organisms, is the most commonly spun extracellular fibrous protein. It comprises polypeptide chains with regions which can crystallize and regions which are predominantly amorphous. The polymer chains in the crystalline regions form anti-parallel pleated sheet structures with an orthorhombic unit cell. Dragline silk is a structural material produced by a variety of spiders. It has been genetically tailored to meet a specific purpose. Dragline silk exhibits high extensibility and tensile strength approaching that of high-strength synthetic fibers. The specific energy to break it can exceed some steels and synthetic fibers. Samples of Nephila clavipes (golden orb-weaver) dragline silk were extracted from live specimens and examined with a series of experimental techniques including optical, scanning electron, and atomic force microscopy, wide and small angle X-ray diffraction and birefringence compensation. Computer modeling of the mechanical properties of the crystallite was also performed. An assortment of features at a variety of length scales was observed by microscopy. These occur on both the as-spun and abraded silk surfaces. The silk was observed to undergo large deformations without evidence of failure, suggesting the absence of a microfibrillar structure. There was no conclusive evidence for either a microfibrillar or a skin core structure. Meridional and equatorial SAXD peaks were observed at Bragg spacings of 79 AA and 250 AA, respectively. Analysis of the WAXD patterns indicated that the silk belongs in Warwicker's category 3b and that the minimum dimensions of the crystals are approximately 38 AA in the molecular direction and 16 x 23 AA in the transverse directions. The crystal modulus was determined with WAXD to be 16.7 GPa, applying the assumption of uniform stress. This is lower than the 200 GPa modulus calculated with molecular modeling. These results and other factors indicate the inapplicability of the widely used assumption of uniform stress. The X-ray data show increased alignment of the crystals along the fiber axis with stress. The birefringence of the silk fibers increases monotonically to failure which indicates increasing molecular alignment. The change in birefringence appears to be governed by the amorphous contribution.

Mahoney, David Vincent

264

Release and cellular acceptance of multiple drugs loaded silk fibroin particles.  

PubMed

In this article, silk fibroin particles with average diameter of 980 nm were fabricated via self assembly. Exceptional loading efficiency and release patterns of hydrophobic and protein drugs were observed. Furthermore, smoother release patterns were observed with increase loading of the hydrophobic and protein model drugs, only about 23% FITC-BSA and 34% RhB were released from the silk particles at their highest corresponding loading in 50 days. Most importantly, osteoblasts' viability was augmented during co-culture with silk fibroin particles, as shown by Alamar Blue assay. Attachment of the particles and delivery of model drugs to cells were confirmed by fluorescence images and flow cytometry. Hence, the silk fibroin particles could be potential biomaterial for application in controlled release and pharmaceutics. PMID:21920418

Shi, Pujiang; Goh, James C H

2011-09-06

265

Structure modifications induced in silk fibroin by enzymatic treatments. A Raman study  

NASA Astrophysics Data System (ADS)

Raman spectroscopy was used to investigate various enzyme-catalyzed reactions onto silk fibroin, i.e. the biodegradation of Tussah (Antheraea pernyi) silk fibroin films by a proteolytic enzyme, the oxidation of domestic (Bombyx mori) silk fibroin by mushroom tyrosinase and the subsequent grafting of chitosan onto oxidized silk. The spectra of Tussah silk fibroin films exposed to a bacterial protease for different times demonstrated that the cleavage of sensitive peptide bonds in the amorphous glycine-rich domains resulted in the loss of various amino acid residues (Tyr, Trp, Asp, etc.). The bands attributed to the crystalline alanine-rich sequences increased in intensity, and the ?-sheet molecular conformation was not affected by biodegradation. Following oxidation with mushroom tyrosinase, the tyrosine bands of Bombyx mori fibroin decreased in intensity but did not disappear. The increase of the I853/I829 intensity ratio indicated that the Tyr residues not accessible to the enzyme were located in a strongly hydrophobic environment. Raman spectroscopy provided evidence that chitosan was effectively grafted onto oxidized silk, probably via the Schiff-base mechanism, as shown by the behavior of the imine band at about 1646 cm-1. Grafting chitosan onto silk fibroin resulted in a ?-sheet?random coil conformational transition of the protein component in the bioconjugated product.

Monti, Patrizia; Freddi, Giuliano; Sampaio, Sandra; Tsukada, Masuhiro; Taddei, Paola

2005-06-01

266

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin, gelatin and hydroxyapatite.  

PubMed

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin/Thai silk fibroin (CGSF), hydroxyapatite/Thai silk fibroin (SF4), and hydroxyapatite/conjugated gelatin/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen leaching, dehydrothermal/chemical crosslinking and an alternate soaking technique for mineralization. In vitro biodegradation in collagenase showed that CGSF scaffolds had the slowest biodegradability, due to the double crosslinking by dehydrothermal and chemical treatments. The hydroxyapatite deposited from alternate soaking separated from the surface of the protein scaffolds when immersed in collagenase. From in vivo biodegradation studies, all scaffolds could still be observed after 12 weeks of implantation in subcutaneous tissue of Wistar rats and also following ISO10993-6: Biological evaluation of medical devices. At 2 and 4 weeks of implantation the four types of Thai silk fibroin based-scaffolds were classified as "non-irritant" to "slight-irritant", compared to Gelfoam(®) (control samples). These natural Thai silk fibroin-based scaffolds may provide suitable biomaterials for clinical applications. PMID:20976530

Tungtasana, Hathairat; Shuangshoti, Somruetai; Shuangshoti, Shanop; Kanokpanont, Sorada; Kaplan, David L; Bunaprasert, Tanom; Damrongsakkul, Siriporn

2010-10-07

267

Silk Self-Assembly Mechanisms and Control-From Thermodynamics to Kinetics  

PubMed Central

Silkworms and spiders generate fibres that exhibit high strength and extensibility. The underlying mechanisms involved in processing silk proteins into fiber form remain incompletely understood, resulting in the failure to fully recapitulate the remarkable properties of native fibers in vitro from regenerated silk solutions. In the present study, the extensibility and high strength of regenerated silks were achieved by mimicking the natural spinning process. Conformational transitions inside micelles, followed by aggregation of micelles and their stabilization as they relate to the metastable structure of silk are described. Subsequently, the mechanisms to control the formation of nanofibrous structures were elucidated. The results clarify that the self-assembly of silk in aqueous solution is a thermodynamically driven process where kinetics also play a key role. Four key factors, molecular mobility, charge, hydrophilic interactions and concentration underlie the process. Adjusting these factors can balance nanostructure and conformational composition, and be used to achieve silk-based materials with properties comparable to native fibers. These mechanisms suggest new directions to design silk-based multifunctional materials.

Lu, Qiang; Zhu, Hesun; Zhang, Cencen; Zhang, Feng; Zhang, Bing; Kaplan, David L.

2012-01-01

268

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides.  

PubMed

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L; Egles, Christophe

2012-09-10

269

Bacterial cellulose nanocrystals-embedded silk nanofibers.  

PubMed

Nanofibrous Bacterial cellulose nanocrystals (BCNs)-embedded silk fibroin were successfully fabricated using electrospinning. The morphology, structure and mechanical properties of the silk fibroin nanofibers were investigated at various BCNs concentrations from 0 to 7 wt%. SEM, TEM and XRD analyses were conducted to confirm the incorporation of the BCNs in the electrospun silk fibroin nanofibers. The average diameter of the silk fibroin/BCNs nanofibers increased from 230 to 430 nm according to the increasing of the BCNs ratio due to the rising solute content. The FT-IR spectra confirmed the conformational transition of the silk fibroin, from a random coil to a beta-sheet structure, which shows the enhanced mechanical properties of silk fibroin based nanofibers even with small amounts of the BCNs. Moreover, it was observed that the Young's modulus of the silk fibroin/BCNs nanofibers unexpectedly increased with the formation of BCNs with a percolation structure at a concentration between 3 and 5 wt%. PMID:22966722

Park, Doo Jin; Choi, Youngeun; Heo, Semi; Cho, Se Youn; Jin, Hyung-Joon

2012-07-01

270

Degradation Mechanism and Control of Silk Fibroin  

PubMed Central

Controlling the degradation process of silk is an important and interesting subject in biomaterials field. In the present study, silk fibroin films with different secondary conformations and nanostructures were used to study the degradation behavior. Silk fibroin films with highest ?-sheet content achieved highest degradation rate, different from the previous studies. A new degradation mechanism revealed that degradation behavior of silk fibroin was related to not only crystal content, but also hydrophilic interaction and crystal-noncrystal alternant nanostructures. The hydrophilic blocks of silk were firstly degraded. Then, the hydrophobic crystal blocks which were formerly surrounded and immobilized by hudrophilic blocks, became free particles and moved into solution. Based on the mechanism, which enables the process more controllable and flexible, controlling the degradation behavior of silk fibroin without sacrificing other performances such as mechanical or hydrophilic properties become feasible, and this would greatly expand the applications of silk as a biomedical material.

Lu, Qiang; Zhang, Bing; Li, Mingzhong; Zuo, Baoqi; Kaplan, David L.; Huang, Yongli; Zhu, Hesun

2012-01-01

271

Identification and synthesis of novel biomaterials based on spider structural silk fibers  

NASA Astrophysics Data System (ADS)

The diversity in function and mechanical behavior of spider silks, and the ability to produce these silks recombinantly, have tremendous potential in creating a new class of biomimetic materials. Here we investigate the structural and mechanical properties of pyriform silks from the golden orb-weaver, Nephila clavipes. Nanoscale indentation measurements using atomic force microscopy on natural pyriform silk suggests that this biomaterial has high toughness that may be suitable for dissipating high amounts of mechanical energy. We also observed the occurrence of highly organized nanocrystals within the pyriform silk fibers that may contribute to the remarkable energy dissipation capability of these silks. It has been demonstrated that poly-(Gly-Ala) and poly-Ala stretches within the internal block repeat modules of dragline silk fibroins form nanocrystals, and these nanocrystalline structures may be responsible for the high extensibility of the dragline silks. In contrast, amino acid sequence analysis shows that PySp2 does not contain the same motifs. In the absence of poly-(Gly-Ala) and poly-Ala repeats, we hypothesized that PySp2 contains new protein motifs sufficient to polymerize into functional structures. To investigate the functional contributions of these novel motifs during pyriform fiber formation, we expressed different recombinant PySp2 fibroins with various segments spanning its block repeat units. We demonstrate that PySp2 recombinant proteins with the Pro-rich sub-block domain (PXP motifs, where X= sub-set of the amino acids A, L, or R) and/or the Ser + Gln + Ala-rich sub-block domain (QQSSVAQS motifs) are sufficient for artificial fiber formation. Moreover, we show that recombinant PySp2 proteins that contain a single block repeat unit can self-assemble into foam-like nanostructures. Collectively, our findings support the use of PySp2 recombinant proteins for a wide range of biomimetic materials with morphologies ranging from fibers to porous structures.

Hsia, Yang; Gnesa, Eric; Tang, Simon; Jeffery, Felicia; Geurts, Paul; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-11-01

272

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration.  

PubMed

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes. PMID:19414015

Shaik, Haq Abdul; Sehnal, Frantisek

2009-05-18

273

Atomic force microscopy of orb-spider-web-silks to measure surface nanostructuring and evaluate silk fibers per strand  

Microsoft Academic Search

Atomic force microscopy (AFM) study is used to measure the surface topology and roughness of radial and capture spider silks on the micro- and nanoscale. This is done for silks of the orb weaver spider Argiope keyserlingi. Capture silk has a surface roughness that is five times less than that for radial silk. The capture silk has an equivalent flatness

D. M. Kane; N. Naidoo; G. R. Staib

2010-01-01

274

A conserved spider silk domain acts as a molecular switch that controls fibre assembly.  

PubMed

A huge variety of proteins are able to form fibrillar structures, especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal and carboxy-terminal domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation initiated by changes in ionic composition and mechanical stimuli known to align the repetitive sequence elements and promote beta-sheet formation. However, despite recent structural data, little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre. PMID:20463741

Hagn, Franz; Eisoldt, Lukas; Hardy, John G; Vendrely, Charlotte; Coles, Murray; Scheibel, Thomas; Kessler, Horst

2010-05-13

275

Judaism and the Silk Route.  

ERIC Educational Resources Information Center

Demonstrates that the Judeans traveled along the Ancient Silk Route. Discusses the Iranian influence on the formation of Jewish religious ideas. Considers the development of Jewish trade networks, focusing on the Radanites (Jewish traders), the Jewish presence in the Far East, and the survival of Judaism in central Asia. (CMK)

Foltz, Richard

1998-01-01

276

Ion Electrodiffusion Governs Silk Electrogelation.  

PubMed

Silk electrogelation involves the transition of an aqueous silk fibroin solution to a gel state (E-gel) in the presence of an electric current. The process is based on local pH changes as a result of water electrolysis - generating H(+) and OH(-) ions at the (+) and (-) electrodes, respectively. Silk fibroin has a pI=4.2 and when local pHsilk-solution)~10, a finite-element ion electrodiffusion model was developed. The model relies on electrodiffusion of the generated H(+) and OH(-) ions. Initially, inputs into the model were the measured E-gel and voltage curves. The governing ion electrodiffusion equations were solved and the calculated pH matched the experimental pH profile, indicating that ion electrodiffusion dictates local pH changes and E-gel growth. Furthermore, the model predicted the constant currents (2mA and 3mA) necessary for two hypothetical E-gel growth curves and these results were then validated experimentally. The model thus shows how ion electrodiffusion governs the electrogelation process and also provides predictable outcomes for fundamental and practical E-gel applications. PMID:22822409

Kojic, Nikola; Panzer, Matthew J; Leisk, Gary G; Raja, Waseem K; Kojic, Milos; Kaplan, David L

2012-05-28

277

Review structure of silk by raman spectromicroscopy: from the spinning glands to the fibers.  

PubMed

Raman spectroscopy has long been proved to be a useful tool to study the conformation of protein-based materials such as silk. Thanks to recent developments, linearly polarized Raman spectromicroscopy has appeared very efficient to characterize the molecular structure of native single silk fibers and spinning dopes because it can provide information relative to the protein secondary structure, molecular orientation, and amino acid composition. This review will describe recent advances in the study of the structure of silk by Raman spectromicroscopy. A particular emphasis is put on the spider dragline and silkworm cocoon threads, other fibers spun by orb-weaving spiders, the spinning dope contained in their silk glands and the effect of mechanical deformation. Taken together, the results of the literature show that Raman spectromicroscopy is particularly efficient to investigate all aspects of silk structure and production. The data provided can lead to a better understanding of the structure of the silk dope, transformations occurring during the spinning process, and structure and mechanical properties of native fibers. PMID:21882171

Lefèvre, Thierry; Paquet-Mercier, François; Rioux-Dubé, Jean-François; Pézolet, Michel

2011-08-31

278

Molecular and nanostructural mechanisms of deformation, strength and toughness of spider silk fibrils.  

PubMed

Spider dragline silk is one of the strongest, most extensible and toughest biological materials known, exceeding the properties of many engineered materials including steel. Silk features a hierarchical architecture where highly organized, densely H-bonded beta-sheet nanocrystals are arranged within a semiamorphous protein matrix consisting of 3(1)-helices and beta-turn protein structures. By using a bottom-up molecular-based approach, here we develop the first spider silk mesoscale model, bridging the scales from Angstroms to tens to potentially hundreds of nanometers. We demonstrate that the specific nanoscale combination of a crystalline phase and a semiamorphous matrix is crucial to achieve the unique properties of silks. Our results reveal that the superior mechanical properties of spider silk can be explained solely by structural effects, where the geometric confinement of beta-sheet nanocrystals, combined with highly extensible semiamorphous domains, is the key to reach great strength and great toughness, despite the dominance of mechanically inferior chemical interactions such as H-bonding. Our model directly shows that semiamorphous regions govern the silk behavior at small deformation, unraveling first when silk is being stretched and leading to the large extensibility of the material. Conversely, beta-sheet nanocrystals play a significant role in defining the mechanical behavior of silk at large-deformation. In particular, the ultimate tensile strength of silk is controlled by the strength of beta-sheet nanocrystals, which is directly related to their size, where small beta-sheet nanocrystals are crucial to reach outstanding levels of strength and toughness. Our results and mechanistic insight directly explain recent experimental results, where it was shown that a significant change in the strength and toughness of silk can be achieved solely by tuning the size of beta-sheet nanocrystals. Our findings help to unveil the material design strategy that enables silk to achieve superior material performance despite simple and inferior material constituents. This concept could lead to a new materials design paradigm, where enhanced functionality is not achieved using complex building blocks but rather through the utilization of simple repetitive constitutive elements arranged in hierarchical structures from nano to macro. PMID:20518518

Nova, Andrea; Keten, Sinan; Pugno, Nicola M; Redaelli, Alberto; Buehler, Markus J

2010-07-14

279

Spider silk has an ice nucleation activity  

NASA Astrophysics Data System (ADS)

Several ice nucleating substances have been identified, which exist in vivo or can be extracted from biological materials. Spider silk, which has a strong ability for water condensation, has also been found to possess an ice nucleation activity. The freezing temperature of water droplets was higher in the presence than in the absence of spider silk. Moreover, by means of environmental scanning electron microscopy, it was observed that the activity is not due to foreign matter attached to the silk but to the silk fibroin itself.

Murase, Norio; Ruike, Masatoshi; Matsunaga, Naoki; Hayakawa, Masahiro; Kaneko, Yoshinori; Ono, Yuzo

2001-03-01

280

Biosynthesis of insulin-silk fibroin nanoparticles conjugates and in vitro evaluation of a drug delivery system  

Microsoft Academic Search

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When it was dissolved in highly concentrated CaCl2 solution and then the mixture of the protein and salt was subjected to desalting treatments for long time in flowing water,\\u000a the resulting liquid silk was water-soluble polypeptides with different molecular masses, ranging from 8 to 70 kDa. When the\\u000a liquid

Hai-Bo Yan; Yu-Qing Zhang; Yong-Lei Ma; Li-Xia Zhou

2009-01-01

281

Plasticity in Major Ampullate Silk Production in Relation to Spider Phylogeny and Ecology  

Microsoft Academic Search

Spider major ampullate silk is a high-performance biomaterial that has received much attention. However, most studies ignore plasticity in silk properties. A better understanding of silk plasticity could clarify the relative importance of chemical composition versus processing of silk dope for silk properties. It could also provide insight into how control of silk properties relates to spider ecology and silk

Cecilia Boutry; Milan ?ezá?; Todd Alan Blackledge

2011-01-01

282

Silk-Fibrin/Hyaluronic Acid Composite Gels for Nucleus Pulposus Tissue Regeneration  

PubMed Central

Scaffold designs are critical for in vitro culture of tissue-engineered cartilage in three-dimensional environments to enhance cellular differentiation for tissue engineering and regenerative medicine. In the present study we demonstrated silk and fibrin/hyaluronic acid (HA) composite gels as scaffolds for nucleus pulposus (NP) cartilage formation, providing both biochemical support for NP outcomes as well as fostering the retention of size of the scaffold during culture due to the combined features of the two proteins. Passage two (P2) human chondrocytes cultured in 10% serum were encapsulated within silk-fibrin/HA gels. Five study groups with fibrin/HA gel culture (F/H) along with varying silk concentrations (2% silk gel only, fibrin/HA gel culture with 1% silk [F/H+1S], 1.5% silk [F/H+1.5S], and 2% silk [F/H+2S]) were cultured in serum-free chondrogenic defined media (CDM) for 4 weeks. Histological examination with alcian blue showed a defined chondrogenic area at 1 week in all groups that widened homogenously until 4 weeks. In particular, chondrogenic differentiation observed in the F/H+1.5S had no reduction in size throughout the culture period. The results of biochemical and molecular biological evaluations supported observations made during histological examination. Mechanical strength measurements showed that the silk mixed gels provided stronger mechanical properties for NP tissue than fibrin/HA composite gels in CDM. This effect could potentially be useful in the study of in vitro NP tissue engineering as well as for clinical implications for NP tissue regeneration.

Park, Sang-Hyug; Cho, Hongsik; Gil, Eun Seok; Mandal, Biman B.; Min, Byoung-Hyun

2011-01-01

283

Effect of ?-sheet crystalline content on mass transfer in silk films  

PubMed Central

The material properties of silk are favorable for drug delivery due to the ability to control material structure and morphology under ambient, aqueous processing conditions. Mass transport of compounds with varying physical-chemical characteristics was studied in silk fibroin films with control of ?-sheet crystalline content. Two compounds, vitamin B12 and fluorescein isothiocynate (FITC) labeled lysozyme were studied in a diffusion apparatus to determine transport through silk films. The films exhibited size exclusion phenomenon with permeability coefficients with contrasting trends with increases in ?-sheet crystallinity. The size exclusion phenomenon observed with the two model compounds was characterized by contrasting trends in permeability coefficients of the films as a function of ?-sheet crystallinity. The diffusivity of the compounds was examined in the context of free volume theory. Apart from the ?-sheet crystallinity, size of the compound and its interactions with silk influenced mass transfer. Diffusivity of vitamin B12 was modeled to define a power law relationship with ?-sheet crystallinity. The results of the study demonstrate that diffusion of therapeutic agents though silk fibroin films can be directed to match a desired rate by modulating secondary structure of the silk proteins.

Karve, Kiran A.; Gil, Eun Seok; McCarthy, Stephen P.; Kaplan, David L.

2011-01-01

284

Effect of silicon on the formation of silk fibroin/calcium phosphate composite.  

PubMed

The silk fibroin/calcium phosphate composites were prepared by adding the different amount of Na(2)SiO(3) to assess the effect of silicon on the HA (hydroxyapatite) formation in the composites. FTIR and XRD results suggested that the inorganic phase was constituted mainly by the amorphous DCPD (dicalcium phosphate dehydrate), a precursor of HA in the bone mineral, when the composites were prepared at the final Na(2)SiO(3) concentration lower than 0.008%. Otherwise, HA was formed as the predominant one in the as-prepared composite, accompanied with a conformational transition in the organic phase of silk fibroin protein from silk I (alpha-helix and/or polyglycine II (3(1)-helix) conformations) to silk II (antiparallel beta-sheet conformation). SEM images showed the different morphologies with the samples, i.e., sheet-like crystals in the composites prepared at a low Na(2)SiO(3) concentration and rod-like bundles in other composites. The rod-like bundles were connected together to form the porous network, due to the fact that the HA crystals grew with the aggregation of silk fibroin, and further accreted onto the silk fibroin fibrils. TG curves indicated that the composites prepared with a certain amount of additional SiO (3) (2-) had the higher thermal stability because of its high molecular orientation and crystallinity, and high water-holding capacity due to the porous microstructure. PMID:17619986

Li, Li; Wei, Ke-Min; Lin, Feng; Kong, Xiang-Dong; Yao, Ju-Ming

2007-07-10

285

Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.  

PubMed

Previously, we established a method to produce recombinant proteins (r-proteins) in cocoons of germline transgenic silkworms, and showed that a step(s) in post-transcription processes was rate-limiting in obtaining a high yield of r-proteins. In this study, we examined whether the 5'-untranslated region (5'-UTR) of the polyhedrin gene (pol) of nucleopolyhedrovirus (NPV) has a translational enhancer activity in the r-protein expression by middle silk gland (MSG) cells of silkworm Bombyx mori (Bm). Sericin 1 gene (ser1) promoter-driven transformation vectors were constructed in which pol5'-UTRs of NPVs isolated from four different species, Bm, Spodoptera frugiperda, Ectropis oblique, and Malacosoma neustria, were each placed upstream of a reporter gene. Transient expression assays in MSGs showed that these pol5'-UTRs all enhanced the protein expression of reporter genes, and the pol5'-UTR of Bm NPV (pol5'-UTR/Bm) was the most effective among them. Thus, transgenic silkworms were generated, which bore the ser1 promoter-driven His-tagged secretory EGFP (sEGFP-His) gene under the control of pol5'-UTR/Bm. The synthesis of sEGFP-His proteins in MSGs of the transgenic worms was approximately 1.5-fold higher than that in those bearing null vectors. However, its mRNA expression levels were 67% of the control worms, indicating that the pol5'-UTR/Bm specifically enhanced the translational level. In conclusion, pol5'-UTR/Bm increased the yield of r-protein production in transgenic silkworms by enhancing the translational activity and this 5'-UTR could be useful for the mass production of r-proteins in germline transgenic silkworms. PMID:18640598

Iizuka, Masashi; Tomita, Masahiro; Shimizu, Katsuhiko; Kikuchi, Yutaka; Yoshizato, Katsutoshi

2008-06-01

286

Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus.  

PubMed

Spiders spin high performance threads that have diverse mechanical properties for specific biological applications. To better understand the molecular mechanism by which spiders anchor their threads to a solid support, we solubilized the attachment discs from black widow spiders and performed in-solution tryptic digests followed by MS/MS analysis to identify novel peptides derived from glue silks. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and cDNA library screening, we isolated a novel member of the silk gene family called pysp1 and demonstrate that its protein product is assembled into the attachment disc silks. Alignment of the PySp1 amino acid sequence to other fibroins revealed conservation in the non-repetitive C-terminal region of the silk family. MS/MS analysis also confirmed the presence of MaSp1 and MaSp2, two important components of dragline silks, anchored within the attachment disc materials. Characterization of the ultrastructure of attachment discs using scanning electron microscopy studies support the localization of PySp1 to small diameter fibers embedded in a glue-like cement, which network with large diameter dragline silk threads, producing a strong, adhesive material. Consistent with elevated PySp1 mRNA levels detected in the pyriform gland, MS analysis of the luminal contents extracted from the pyriform gland after tryptic digestion support the assertion that PySp1 represents one of the major constituents manufactured in the pyriform gland. Taken together, our data demonstrate that PySp1 is spun into attachment disc silks to help affix dragline fibers to substrates, a critical function during spider web construction for prey capture and locomotion. PMID:19666476

Blasingame, Eric; Tuton-Blasingame, Tiffany; Larkin, Leah; Falick, Arnold M; Zhao, Liang; Fong, Justine; Vaidyanathan, Veena; Visperas, Anabelle; Geurts, Paul; Hu, Xiaoyi; La Mattina, Coby; Vierra, Craig

2009-08-07

287

Nanocomposite gold-silk nanofibers  

PubMed Central

Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNFseed). Following gold reduction, there was a two-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFsAu). The Young’s modulus of the SNFsAu was almost 70% higher than that of SNFs. SNFsAu were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNFAu had a more than two-fold larger cell area compared to the cells cultured on bare SNFs; SNFAu also increase cell size. We suggest that this approach can be used to alter the cell-material interface in tissue engineering and other applications.

Cohen-Karni, Tzahi; Jeong, Kyung Jae; Tsui, Jonathan H.; Reznor, Gally; Mustata, Mirela; Wanunu, Meni; Graham, Adam; Marks, Carolyn; Bell, David C.; Langer, Robert S.; Kohane, Daniel S.

2012-01-01

288

Secrets of the Silk Road  

NSDL National Science Digital Library

Secrets of the Silk Road is another worthy addition to the growing body of Web-based information about the Silk Road (see, for example, The International Dunhuang Project, founded in 1998 and mentioned in several Scout Reports), launched by the Bowers Museum of Santa Ana, California and presented by University of Pennsylvania Museum of Archaeology and Anthropology. This site includes artifacts documenting the history of the Silk Road in the vast Tarim Basin in the Far Western Xinjiang Uyghur Autonomous Region of China. Three well-preserved mummies from the Tarim Basin have been brought to the United States for the exhibition, along with lavish grave goods. Visitors to the website can view images of "The Beauty of Xiaohe," a female mummy from 1800-1500 BCE and other treasures ranging from a Chrysanthemum Shaped Dessert from the 5th-3rd century BCE, to gold objects such as mask from 5th-6th century CE or the Gold Plaque with Lion Design, 5th-3rd Century BCE.

289

Ion Electrodiffusion Governs Silk Electrogelation  

PubMed Central

Silk electrogelation involves the transition of an aqueous silk fibroin solution to a gel state (E-gel) in the presence of an electric current. The process is based on local pH changes as a result of water electrolysis – generating H+ and OH? ions at the (+) and (?) electrodes, respectively. Silk fibroin has a pI=4.2 and when local pH

Kojic, Nikola; Panzer, Matthew J.; Leisk, Gary G.; Raja, Waseem K.; Kojic, Milos; Kaplan, David L.

2012-01-01

290

Total x-ray scattering of spider dragline silk.  

PubMed

Total x-ray scattering measurements of spider dragline silk fibers from Nephila clavipes, Argiope aurantia, and Latrodectus hesperus all yield similar structure factors, with only small variations between the different species. Wide-angle x-ray scattering from fibers orientated perpendicular to the beam shows a high degree of anisotropy, and differential pair distribution functions obtained by integrating over wedges of the equatorial and meridian planes indicate that, on average, the majority (95%) of the atom-atom correlations do not extend beyond 1 nm. Futhermore, the atom-atom correlations between 1 and 3 nm are not associated with the most intense diffraction peaks at Q=1-2 Å(-1). Disordered molecular orientations along the fiber axis are consistent with proteins in similar structural arrangements to those in the equatorial plane, which may be associated with the silk's greater flexibility in this direction. PMID:22680907

Benmore, C J; Izdebski, T; Yarger, J L

2012-04-24

291

Development and characterization of silk fibroin coated quantum dots  

NASA Astrophysics Data System (ADS)

Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs) has seen them find wider acceptance as a tool in biomedical research labs. As produced, high quality QDs, synthesized by high temperature organometallic synthesis, are coated with a hydrophobic ligand. Therefore, they must be further processed to be soluble in water and to be made biocompatible. To accomplish this, the QDs are generally coated with a synthetic polymer (eg. block copolymers) or the hydrophobic surface ligands exchanged with hydrophilic material (eg. thiols). Advances in this area have enabled the QDs to experience a smooth transition from being simple inorganic fluorophores to being smart sensors, which can identify specific cell marker proteins and help in diagnosis of diseases such as cancer. In order to improve the biocompatibility and utility of the QDs, we report the development of a procedure to coat QDs with silk fibroin, a fibrous crystalline protein extracted from Bombyx Mori silkworm. Following the coating process, we characterize the size, quantum yield and two-photon absorption cross section of the silk coated QDs. Additionally, the results of biocompatibility studies carried out to compare the properties of these QD-silks with conventional QDs are presented. These natural polymer coatings on QDs could enhance the intracellular delivery and enable the use of these nanocrystals as an imaging tool for studying subcellular machinery at the molecular level.

Nathwani, B. B.; Needham, C.; Mathur, A. B.; Meissner, K. E.

2008-03-01

292

Structural Transitions Induced by a Recombinant Methionine-Trigger in Silk Spidroin  

NASA Astrophysics Data System (ADS)

Control of beta sheet formation is an important factor in the understanding and prediction of structural transitions and protein folding. In genetically engineered silk proteins this control has been achieved using oxidative triggers. A genetically engineered variant of a spider silk protein, and a peptide analog, based on the consensus sequence of Nephila clavipes dragline silk, were modified to include methionines flanking the beta sheet forming polyalanine regions. These methionines could be selectively reduced and oxidized, altering the bulkiness and charge of the sulfhydryl group to control beta sheet formation by steric hindrance. Biophysical characterization and monitoring of structural transitions and intermediates were accomplished through attenuated total reflectance infrared spectroscopy (ATR-IR) for solution state structures in both oxidized and reduced forms. For solid state structural characterization, IR microscopy and reflectance IR experiments were performed. Electron diffraction data as well as circular dichroism studies provide structural corroboration for all experiments in which reproducible sample preparation was achieved.

Wilson, Donna; Winkler, Stefan; Valluzzi, Regina; Kaplan, David

2000-03-01

293

How to visualize the spider mite silk?  

Microsoft Academic Search

Brightener 28 ABSTRACT Tetranychus urticae (Acari: Tetranychidae) is a phytophagous mite that forms colonies of several thousand individuals. Like spiders, every individual produces abundant silk strands and is able to construct a common web for the entire colony. Despite the importance of this silk for the biology of this worldwide species, only one previous study suggested how to visualize it.

G. Clotuche; G. Le Goff; A-C Mailleux; J-L Deneubourg; C. Detrain; T. Hance

2009-01-01

294

Seljuk Muqarnas along the Silk Road  

Microsoft Academic Search

The film Seljuk Muqarnas along the Silk Road gives an overview of muqarnas, stalactite vaults, in Seljuk style architecture (1038-1194). The muqarnas are located in portals and niches of car- avansaraies, madrassas and mosques. Starting with the Sultan Han near Kayseri we follow the Silk Road westward till Konya and finally show the Arslanhane Camii in Ankara. Video recordings alternate

Silvia Harmsen; Daniel Jungblut; Susanne Kromker

295

Silk: the original liquid crystalline polymer  

Microsoft Academic Search

The word ‘silk’ brings to the mind a kaleidoscope of images: silkworm culture in the villages of China, camel trains on the ancient silk road of Asia, the finery of the mediaeval courts, the workshops of Huguenot refugees in Spitalfields and Macclesfield, Victorian velvet and chiffon mills, the parachutes of Spitfire pilots (and the linings of their battledress), QCs' robes,

John E. Lydon

2004-01-01

296

Spinning solution composition, process for producing regenerated silk fiber using the composition, and regenerated silk fiber produced by the process  

US Patent & Trademark Office Database

A spinning solution composition which provides regenerated silk fiber that exhibits a strength and elongation close to those of natural silk fiber, is rapidly degradable in vivo, and can be caused to have ability to gradually release a drug to prevent inflammation; a process for producing such regenerated silk fiber by use of the composition; and regenerated silk fiber obtained through the process.

2013-01-08

297

Structure and dynamics of aromatic residues in spider silk: 2D carbon correlation NMR of dragline fibers.  

PubMed

The structure of aromatic residues in spider silk has remained relatively uncharacterized. This is primarily due to the relatively low abundance of aromatic residues in dragline spider silk. NMR characterization has been further hindered by the inability to (13)C-isotopically enrich these amino acids in the silk effectively. In the present contribution, it is shown that feeding spiders an aqueous solution of [U-(13)C/(15)N]-l-phenylalanine (13)C-enriches Tyr to a level of 20-30% in dragline spider silk. This enables the collection of 2D through-bond (13)C double quantum/single quantum (DQ/SQ) correlation spectra with the refocused INADEQUATE solid-state NMR pulse sequence. These 2D spectra provide the complete unambiguous assignment of the Tyr resonances in dragline silks from two spider species: N. clavipes and A. aurantia. Additionally, weak resonances are detected for Phe in A. aurantia dragline silk that is present at levels <1%. The conformation dependence of the C(alpha), C(beta), and carbonyl chemical shifts show that Tyr and Phe present in the GGX and GPGXX motifs in the spider silk proteins MaSp1 and MaSp2 are in disordered helical structures and not incorporated in the beta-sheet domains. PMID:19894709

Izdebski, Thomas; Akhenblit, Paul; Jenkins, Janelle E; Yarger, Jeffery L; Holland, Gregory P

2010-01-11

298

Mechanics and Morphology of Silk Drawn from Anesthetized Spiders  

NASA Astrophysics Data System (ADS)

CO2 and N2 anesthetized Nephila spiders produced dragline silk with mechanical properties that differed from control silk as a function of time under anesthesia. Silk from CO2 spiders had a significantly lower breaking strain and breaking energy, significantly higher initial modulus, and marginally lower breaking stress. At the onset of anesthesia the silk diameter became highly variable. During deep anesthesia silk either became thinner or retained cross-section but fibrillated.

Madsen, B.; Vollrath, F.

299

Mandibular Repair in Rats with Premineralized Silk Scaffolds and BMP-2-modified bMSCs  

PubMed Central

Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.

Jiang, Xinquan; Zhao, Jun; Wang, Shaoyi; Sun, Xiaojuan; Zhang, Xiuli; Chen, Jake; Kaplan, David L.; Zhang, Zhiyuan

2010-01-01

300

Gel spinning of silk tubes for tissue engineering  

PubMed Central

Tubular vessels for tissue engineering are typically fabricated using a molding, dipping, or electrospinning technique. While these techniques provide some control over inner and outer diameters of the tube, they lack the ability to align the polymers or fibers of interest throughout the tube. This is an important aspect of biomaterial composite structure and function for mechanical and biological impact of tissue outcomes. We present a novel aqueous process system to spin tubes from biopolymers and proteins such as silk fibroin. Using silk as an example, this method of winding an aqueous solution around a reciprocating rotating mandrel offers substantial improvement in the control of the tube properties, specifically with regard to winding pattern, tube porosity, and composite features. Silk tube properties are further controlled via different post-spinning processing mechanisms such as methanol-treatment, air-drying, and lyophilization. This approach to tubular scaffold manufacture offers numerous tissue engineering applications such as complex composite biomaterial matrices, blood vessel grafts and nerve guides, among others.

Lovett, Michael; Cannizzaro, Christopher; Vunjak-Novakovic, Gordana; Kaplan, David L.

2011-01-01

301

In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for BMP-7 gene delivery.  

PubMed

Abstract Introduction and aims: For a scaffold material to be considered effective and efficient for tissue engineering it must be biocompatible as well as bioinductive. Silk fiber is a natural biocompatible material suitable for scaffold fabrication; however, silk is tissue-conductive and lacks tissue-inductive properties. One proposed method to make the scaffold tissue-inductive is to introduce plasmids or viruses encoding a specific growth factor into the scaffold. In this study, we constructed adenoviruses encoding bone morphogenetic protein-7 (BMP-7) and incorporated these into silk scaffolds. The osteo-inductive and new bone formation properties of these constructs were assessed in vivo in a critical-sized skull defect animal model. Materials and methods: Silk fibroin scaffolds containing adenovirus particles coding BMP-7 were prepared. The release of the adenovirus particles from the scaffolds was quantified by tissue-culture infective dose (TCID50) and the bioactivity of the released viruses was evaluated on human bone marrow mesenchymal stromal cells (BMSCs). To demonstrate the in vivo bone forming ability of the virus-carrying silk fibroin scaffold, the scaffold constructs were implanted into calvarial defects in SCID mice. Results: In vitro studies demonstrated that the virus-carrying silk fibroin scaffold released virus particles over a 3 week period while preserving their bioactivity. In vivo test of the scaffold constructs in critical-sized skull defect areas revealed that silk scaffolds were capable of delivering the adenovirus encoding BMP-7, resulting significantly enhanced new bone formation. Conclusions: Silk scaffolds carrying BMP-7 encoding adenoviruses can effectively transfect cells and enhance both in vitro and in vivo osteogenesis. The findings of this study indicate silk fibroin is a promising biomaterial for gene delivery to repair critical-sized bone defects. PMID:21417715

Zhang, Yufeng; Fan, Wei; Nothdurft, Luke; Wu, Chengtie; Zhou, Yinghong; Crawford, Ross; Xiao, Yin

2011-03-18

302

In vivo Degradation of Three-Dimensional Silk Fibroin Scaffolds  

PubMed Central

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP) have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation has not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well-tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all aqueous process degraded to completion between two and six months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond one year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100–200 µm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.

Wang, Yongzhong; Rudym, Darya D.; Walsh, Ashley; Abrahamsen, Lauren; Kim, Hyeon-Joo; Kim, Hyun Suk; Kirker-Head, Carl; Kaplan, David L.

2011-01-01

303

How to visualize the spider mite silk?  

PubMed

Tetranychus urticae (Acari: Tetranychidae) is a phytophagous mite that forms colonies of several thousand individuals. Like spiders, every individual produces abundant silk strands and is able to construct a common web for the entire colony. Despite the importance of this silk for the biology of this worldwide species, only one previous study suggested how to visualize it. To analyze the web structuration, we developed a simple technique to dye T. urticae'silk on both inert and living substrates. Fluorescent brightener 28 (FB) (Sigma F3543) diluted in different solvents at different concentrations regarding the substrate was used to observe single strands of silk. On glass lenses, a 0.5% dimethyl sulfoxide solution was used and on bean leaves, a 0.1% aqueous solution. A difference of silk deposit was observed depending the substrate: rectilinear threads on glass lenses and more sinuous ones on bean leaves. This visualizing technique will help to carry out future studies about the web architecture and silk used by T. urticae. It might also be useful for the study of other silk-spinning arthropods. PMID:19322898

Clotuche, G; Le Goff, G; Mailleux, A-C; Deneubourg, J-L; Detrain, C; Hance, T

2009-09-01

304

Evaluation of Gel Spun Silk-Based Biomaterials in a Murine Model of Bladder Augmentation  

PubMed Central

Currently, gastrointestinal segments are considered the gold standard for bladder reconstructive procedures. However, significant complications including chronic urinary tract infection, metabolic abnormalities, urinary stone formation, bowel dysfunction, and secondary malignancies are associated with this approach. Biomaterials derived from silk fibroin may represent a superior alternative due their robust mechanical properties, biodegradable features, and processing plasticity. In the present study, we evaluated the efficacy of a gel spun silk-based matrix for bladder augmentation in a murine model. Over the course of 70 d implantation period, H&E and Masson’s trichrome (MTS) analysis revealed that silk matrices were capable of supporting both urothelial and smooth muscle regeneration at the defect site. Prominent uroplakin and contractile protein expression (?-actin, calponin, and SM22?) was evident by immunohistochemical analysis demonstrating maturation of the reconstituted bladder wall compartments. Gel spun silk matrices also elicited a minimal acute inflammatory reaction following 70 d of bladder integration, in contrast to parallel assessments of small intestinal submucosa (SIS) and polyglycolic acid (PGA) matrices which routinely promoted evidence of fibrosis and chronic inflammatory responses. Voided stain on paper analysis revealed that silk augmented animals displayed similar voiding patterns in comparison to non surgical controls by 42 d of implantation. In addition, cystometric evaluations of augmented bladders at 70 d post-op demonstrated that silk scaffolds supported significant increases in bladder capacity, voided volume, and flow rate while maintaining similar degrees of compliance relative to the control group. These results provide evidence for the utility of gel spun silk-based matrices for functional bladder tissue engineering applications.

Mauney, Joshua R.; Cannon, Glenn M.; Lovett, Michael L.; Gong, Edward M.; DiVizio, Dolores; Kaplan, David L.; Adam, Rosalyn M.; Estrada, Carlos R.

2013-01-01

305

Quantitative Research on Brand Elements and Domestic Silk Brand Cognition Based on Silk Products  

Microsoft Academic Search

\\u000a This article designed a brand elements questionnaire combining the brand marketing theory from the point of consumers, analyzed\\u000a the market research data with the structure equation model thought and analysis on SPSS13.0 software. Empirically researched\\u000a the elements of silk brand and cognitive status on main silk products brand of domestic consumer.The results offered reference\\u000a value for positioning China Silk brand,

Rui Shi; Guolian Liu

306

In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds  

PubMed Central

Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell–silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture.

Kim, Hyeon Joo; Park, Sang-Hyug; Durham, Jennah; Gimble, Jeffrey M; Kaplan, David L

2012-01-01

307

Dating silk by capillary electrophoresis mass spectrometry.  

PubMed

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 ?g 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. PMID:21913691

Moini, Mehdi; Klauenberg, Kathryn; Ballard, Mary

2011-09-13

308

Dynamic behaviour of silks: Nature's precision nanocomposites  

NASA Astrophysics Data System (ADS)

Silk is often cited as a material worth imitating, due to its high strength and toughness. In order to produce a synthetic analogue, or enhanced natural version, the microstructural basis of these properties must be understood. Current understanding is that silk deforms through the detachment of nano-scale crystallites, in the manner of a damaged composite. This picture forms the basis for constitutive models, but validation data is limited to low strain-rates. Here we present a programme of research in which high-rate behaviour is studied through ballistic impact experiments. These have been applied to the silk of the Bombyx mori moth, as harvested from cocoons, and to the major ampullate thread of the golden orb weaver spider Nephila edulis. Longitudinal wave-speeds, and air drag coefficients, have been calculated for selected cases. Differences between the response of various silks and a similar synthetic fibre, nylon, are discussed, and future plans are presented.

Drodge, D. R.; Mortimer, B.; Siviour, C. R.; Holland, C.

2012-08-01

309

Spinning an elastic ribbon of spider silk.  

PubMed Central

The Sicarid spider Loxosceles laeta spins broad but very thin ribbons of elastic silk that it uses to form a retreat and to capture prey. A structural investigation into this spider's silk and spinning apparatus shows that these ribbons are spun from a gland homologous to the major ampullate gland of orb web spiders. The Loxosceles gland is constructed from the same basic parts (separate transverse zones in the gland, a duct and spigot) as other spider silk glands but construction details are highly specialized. These differences are thought to relate to different ways of spinning silk in the two groups of spiders. Loxosceles uses conventional die extrusion, feeding a liquid dope (spinning solution) to the slit-like die to form a flat ribbon, while orb web spiders use an extrusion process in which the silk dope is processed in an elongated duct to produce a cylindrical thread. This is achieved by the combination of an initial internal draw down, well inside the duct, and a final draw down, after the silk has left the spigot. The spinning mechanism in Loxosceles may be more ancestral.

Knight, David P; Vollrath, Fritz

2002-01-01

310

Inducing ?-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching.  

PubMed

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of ?-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers. PMID:21574576

An, Bo; Hinman, Michael B; Holland, Gregory P; Yarger, Jeffery L; Lewis, Randolph V

2011-05-24

311

pH-Dependent Anticancer Drug Release from Silk Nanoparticles.  

PubMed

Silk has traditionally been used as a suture material because of its excellent mechanical properties and biocompatibility. These properties have led to the development of different silk-based material formats for tissue engineering and regenerative medicine. Although there have been a small number of studies about the use of silk particles for drug delivery, none of these studies have assessed the potential of silk to act as a stimulus-responsive anticancer nanomedicine. This report demonstrates that an acetone precipitation of silk allows the formation of uniform silk nanoparticles (98 nm diameter, polydispersity index 0.109), with an overall negative surface charge (-33.6 ± 5.8 mV), in a single step. Silk nanoparticles are readily loaded with doxorubicin (40 ng doxorubicin/?g silk) and show pH-dependent release (pH 4.5? 6.0 > 7.4). In vitro studies with human breast cancer cell lines demonstrates that the silk nanoparticles are not cytotoxic (IC50 > 120 ?g mL(-1) ) and that doxorubicin-loaded silk nanoparticles are able to overcome drug resistance mechanisms. Live cell fluorescence microscopy studies show endocytic uptake and lysosomal accumulation of silk nanoparticles. In summary, the pH-dependent drug release and lysosomal accumulation of silk nanoparticles demonstrate the ability of drug-loaded silk nanoparticles to serve as a lysosomotropic anticancer nanomedicine. PMID:23625825

Seib, F Philipp; Jones, Gregory T; Rnjak-Kovacina, Jelena; Lin, Yinan; Kaplan, David L

2013-04-26

312

Silk fibers and silk-producing organs of Harpactea rubicunda (C. L. Koch 1838) (Araneae, Dysderidae).  

PubMed

Scanning electron microscopy and atomic force microscopy were used to study the silk spinning apparatus and silks of Harpactea rubicunda spiders. Three types of silk secretions that are produced by three kinds of silk spinning glands (ampullate, piriform, and pseudaciniform) and released through three types of spigots, were confirmed for both adult and juvenile spiders. Silk secretions for the construction of spider webs for shelter or retreat are produced by the pseudaciniform silk glands. Silk secretions that are released from spigots in the course of web construction are not processed by the legs during the subsequent process of hardening. Pairs of nanofibril bundles seemed to be part of the basic microarchitecture of the web silk fibers as revealed by AFM. These fiber bundles frequently not only overlap one another, but occasionally also interweave. This structural variability may strengthen the spider web. High-resolution AFM scans of individual nanofibrils show a distinctly segmented nanostructure. Each globular segment is ?30-40 nm long along the longitudinal axis of the fiber, and resembles a nanosegment of artificial fibroin described by Perez-Rigueiro et al. (2007). PMID:23034869

Hajer, Jaromír; Malý, Jan; Reháková, Dana

2012-10-04

313

Silk Roads or Steppe Roads? The Silk Roads in World History.  

ERIC Educational Resources Information Center

|Explores the prehistory of the Silk Roads, reexamines their structure and history in the classical era, and explores shifts in their geography in the last one thousand years. Explains that a revised understanding of the Silk Roads demonstrates how the Afro-Eurasian land mass has been linked by networks of exchange since the Bronze Age. (CMK)|

Christian, David

2000-01-01

314

Novel silk fibroin/elastin wound dressings.  

PubMed

Silk fibroin (SF) and elastin (EL) scaffolds were successfully produced for the first time for the treatment of burn wounds. The self-assembly properties of SF, together with the excellent chemical and mechanical stability and biocompatibility, were combined with elastin protein to produce scaffolds with the ability to mimic the extracellular matrix (ECM). Porous scaffolds were obtained by lyophilization and were further crosslinked with genipin (GE). Genipin crosslinking induces the conformational transition from random coil to ?-sheet of SF chains, yielding scaffolds with smaller pore size and reduced swelling ratios, degradation and release rates. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to obtain scaffolds suitable for biological applications. Wound healing was assessed through the use of human full-thickness skin equivalents (EpidermFT). Standardized burn wounds were induced by a cautery and the best re-epithelialization and the fastest wound closure was obtained in wounds treated with 50SF scaffolds; these contain the highest amount of elastin after 6 days of healing in comparison with other dressings and controls. The cytocompatibility demonstrated with human skin fibroblasts together with the healing improvement make these SF/EL scaffolds suitable for wound dressing applications. PMID:22546517

Vasconcelos, Andreia; Gomes, Andreia C; Cavaco-Paulo, Artur

2012-04-27

315

21 CFR 878.5030 - Natural nonabsorbable silk surgical suture.  

Code of Federal Regulations, 2010 CFR

... false Natural nonabsorbable silk surgical suture. 878.5030 Section 878...GENERAL AND PLASTIC SURGERY DEVICES Surgical Devices § 878.5030 Natural nonabsorbable silk surgical suture. (a) Identification....

2009-04-01

316

21 CFR 878.5030 - Natural nonabsorbable silk surgical suture.  

Code of Federal Regulations, 2013 CFR

... false Natural nonabsorbable silk surgical suture. 878.5030 Section 878...GENERAL AND PLASTIC SURGERY DEVICES Surgical Devices § 878.5030 Natural nonabsorbable silk surgical suture. (a) Identification....

2013-04-01

317

21 CFR 878.5030 - Natural nonabsorbable silk surgical suture.  

Code of Federal Regulations, 2010 CFR

... false Natural nonabsorbable silk surgical suture. 878.5030 Section 878...GENERAL AND PLASTIC SURGERY DEVICES Surgical Devices § 878.5030 Natural nonabsorbable silk surgical suture. (a) Identification....

2010-04-01

318

Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae, Theridiidae)  

PubMed Central

With its incredible strength and toughness, spider dragline silk is widely lauded for its impressive material properties. Dragline silk is composed of two structural proteins, MaSp1 and MaSp2, which are encoded by members of the spidroin gene family. While previous studies have characterized the genes that encode the constituent proteins of spider silks, nothing is known about the physical location of these genes. We determined karyotypes and sex chromosome organization for the widow spiders, Latrodectus hesperus and L. geometricus (Araneae, Theridiidae). We then used fluorescence in situ hybridization to map the genomic locations of the genes for the silk proteins that compose the remarkable spider dragline. These genes included three loci for the MaSp1 protein and the single locus for the MaSp2 protein. In addition, we mapped a MaSp1 pseudogene. All the MaSp1 gene copies and pseudogene localized to a single chromosomal region while MaSp2 was located on a different chromosome of L. hesperus. Using probes derived from L. hesperus, we comparatively mapped all three MaSp1 loci to a single region of a L. geometricus chromosome. As with L. hesperus, MaSp2 was found on a separate L. geometricus chromosome, thus again unlinked to the MaSp1 loci. These results indicate orthology of the corresponding chromosomal regions in the two widow genomes. Moreover, the occurrence of multiple MaSp1 loci in a conserved gene cluster across species suggests that MaSp1 proliferated by tandem duplication in a common ancestor of L. geometricus and L. hesperus. Unequal crossover events during recombination could have given rise to the gene copies and could also maintain sequence similarity among gene copies over time. Further comparative mapping with taxa of increasing divergence from Latrodectus will pinpoint when the MaSp1 duplication events occurred and the phylogenetic distribution of silk gene linkage patterns.

Zhao, Yonghui; Ayoub, Nadia A.; Hayashi, Cheryl Y.

2010-01-01

319

Chromosome mapping of dragline silk genes in the genomes of widow spiders (Araneae, Theridiidae).  

PubMed

With its incredible strength and toughness, spider dragline silk is widely lauded for its impressive material properties. Dragline silk is composed of two structural proteins, MaSp1 and MaSp2, which are encoded by members of the spidroin gene family. While previous studies have characterized the genes that encode the constituent proteins of spider silks, nothing is known about the physical location of these genes. We determined karyotypes and sex chromosome organization for the widow spiders, Latrodectus hesperus and L. geometricus (Araneae, Theridiidae). We then used fluorescence in situ hybridization to map the genomic locations of the genes for the silk proteins that compose the remarkable spider dragline. These genes included three loci for the MaSp1 protein and the single locus for the MaSp2 protein. In addition, we mapped a MaSp1 pseudogene. All the MaSp1 gene copies and pseudogene localized to a single chromosomal region while MaSp2 was located on a different chromosome of L. hesperus. Using probes derived from L. hesperus, we comparatively mapped all three MaSp1 loci to a single region of a L. geometricus chromosome. As with L. hesperus, MaSp2 was found on a separate L. geometricus chromosome, thus again unlinked to the MaSp1 loci. These results indicate orthology of the corresponding chromosomal regions in the two widow genomes. Moreover, the occurrence of multiple MaSp1 loci in a conserved gene cluster across species suggests that MaSp1 proliferated by tandem duplication in a common ancestor of L. geometricus and L. hesperus. Unequal crossover events during recombination could have given rise to the gene copies and could also maintain sequence similarity among gene copies over time. Further comparative mapping with taxa of increasing divergence from Latrodectus will pinpoint when the MaSp1 duplication events occurred and the phylogenetic distribution of silk gene linkage patterns. PMID:20877726

Zhao, Yonghui; Ayoub, Nadia A; Hayashi, Cheryl Y

2010-09-21

320

The performance of silk scaffolds in a rat model of augmentation cystoplasty.  

PubMed

The diverse processing plasticity of silk-based biomaterials offers a versatile platform for understanding the impact of structural and mechanical matrix properties on bladder regenerative processes. Three distinct groups of 3-D matrices were fabricated from aqueous solutions of Bombyx mori silk fibroin either by a gel spinning technique (GS1 and GS2 groups) or a solvent-casting/salt-leaching method in combination with silk film casting (FF group). SEM analyses revealed that GS1 matrices consisted of smooth, compact multi-laminates of parallel-oriented silk fibers while GS2 scaffolds were composed of porous (pore size range, 5-50 ?m) lamellar-like sheets buttressed by a dense outer layer. Bi-layer FF scaffolds were comprised of porous foams (pore size, ~400 ?m) fused on their external face with a homogenous, nonporous silk film. Silk groups and small intestinal submucosa (SIS) matrices were evaluated in a rat model of augmentation cystoplasty for 10 weeks of implantation and compared to cystotomy controls. Gross tissue evaluations revealed the presence of intra-luminal stones in all experimental groups. The incidence and size of urinary calculi was the highest in animals implanted with gel spun silk matrices and SIS with frequencies ?57% and stone diameters of 3-4 mm. In contrast, rats augmented with FF scaffolds displayed substantially lower rates (20%) and stone size (2 mm), similar to the levels observed in controls (13%, 2 mm). Histological (hematoxylin and eosin, Masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (?-smooth muscle actin and SM22?) expression within defect sites supported by all matrix groups similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent uroplakin and p63 protein expression in all experimental groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by Fox3-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. In comparison to other biomaterial groups, cystometric analyses at 10 weeks post-op revealed that animals implanted with the FF matrix configuration displayed superior urodynamic characteristics including compliance, functional capacity, as well as spontaneous non voiding contractions consistent with control levels. Our data demonstrate that variations in scaffold processing techniques can influence the in vivo functional performance of silk matrices in bladder reconstructive procedures. PMID:23545287

Seth, Abhishek; Chung, Yeun Goo; Gil, Eun Seok; Tu, Duong; Franck, Debra; Di Vizio, Dolores; Adam, Rosalyn M; Kaplan, David L; Estrada, Carlos R; Mauney, Joshua R

2013-03-29

321

Biomimetic calcium phosphate coatings on recombinant spider silk fibres  

Microsoft Academic Search

Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant

Liang Yang; My Hedhammar; Tobias Blom; Klaus Leifer; Jan Johansson; Pamela Habibovic; Clemens A. van Blitterswijk

2010-01-01

322

Effect of water on the thermal properties of silk fibroin  

Microsoft Academic Search

Silk fibroin films cast from water solution, and containing bound water, are quantitatively studied in this work. First, to obtain the solid and liquid heat capacities of the pure dry silk fibroin, cyclic heat treatment was used to monitor the process of removing the bound water. After water removal, the glass transition of pure non-crystalline silk was observed at 451K

Xiao Hu; David Kaplan; Peggy Cebe

2007-01-01

323

Non-equilibrium Silk Fibroin Adhesives  

PubMed Central

Regenerated silkworm silk solutions formed metastable, soft-solid-like materials (e-gels) under weak electric fields, displaying interesting mechanical characteristics such as dynamic adhesion and strain stiffening. Raman spectroscopy, in situ electric field dynamic oscillatory rheology and polarized optical microscopy indicated that silk fibroin electrogelation involved intermolecular self-assembly of silk molecules into amorphous, micron-scale, micellar structures and the formation of relatively long lifetime, intermicellar entanglement crosslinks. Overall, the electrogelation process did not require significant intramolecular ?-strand or intermolecular ?-sheet formation, unlike silk hydrogels. The kinetics of e-gel formation could be tuned by changing the field strength and assembly conditions, such as silk concentration and solution pH, while e-gel stiffness was partially reversible by removal of the applied field. Transient adhesion testing indicated that the adhesive characteristics of e-gels could at least partially be attributed to a local increase in proton concentration around the positive electrode due to the applied field and surface effects. A working model of electrogelation was described en route to understanding the origins of the adhesive characteristics.

Yucel, Tuna; Kojic, Nikola; Leisk, Gary G.; Lo, Tim J.; Kaplan, David L.

2009-01-01

324

Preparation of uniaxial multichannel silk fibroin scaffolds for guiding primary neurons.  

PubMed

Physical guidance cues have been exploited to stimulate neuron adhesion and neurite outgrowth. In the present study, three-dimensional (3-D) silk fibroin scaffolds with uniaxial multichannels (42-142 ?m in diameter) were prepared by a directional temperature field freezing technique, followed by lyophilization. By varying the initial silk fibroin concentration, the chemical potential and quantity of free water around cylindrical ice crystals could be controlled to control the cross-section morphology of the scaffold channels. Aligned ridges also formed on the inner surface of the multichannels in parallel to the direction of the channels. In vitro, primary hippocampal neurons were seeded in these 3-D silk fibroin scaffolds with uniaxial multichannels of ?120 ?m in diameter. The morphology of the neurons was multipolar and alignment along the scaffold channels was observed. Cell-cell networks and cell-matrix interactions established by newly formed axons were observed after 7 days in culture. These neurons expressed ?-III-tubulin, nerve filament and microtubule-associated protein, while glial fibrillary acidic protein immunofluorescence was barely above background. The ridges on the inner surface of the channels played a critical role in the adhesion and extension of neurons by providing continuous contact guidance. These new 3-D silk scaffolds with uniaxial multichannels provided a favorable microenvironment for the development of hippocampal neurons by guiding axonal elongation and cell migration. PMID:22465574

Zhang, Qiang; Zhao, Yahong; Yan, Shuqin; Yang, Yumin; Zhao, Huijing; Li, Mingzhong; Lu, Shenzhou; Kaplan, David L

2012-03-28

325

Spider Silk-Based Gene Carriers for Tumor Cell-Specific Delivery  

PubMed Central

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 nano-scale 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 non-viral gene delivery.

Numata, Keiji; Reagan, Michaela R; Goldstein, Robert H; Rosenblatt, Michael; Kaplan, David L

2011-01-01

326

Native spider silk as a biological optical fiber  

NASA Astrophysics Data System (ADS)

In this study, we demonstrate the use of eco-friendly native spider silk as an efficient optical fiber in air, highly bent fibers, and physiological liquid. We also integrated the silk filament in a photonic chip made of polymer microstructures fabricated by UV lithography. The molding process is non-destructive for silk and leads to an efficient micro-optical coupling between silk and synthetic optical structures. These optical performances combined with the unique biocompatibility, bioresorbability, flexibility, and tensile strength of silk filaments pave the way for new applications in biological media and for original biophotonic purposes.

Huby, N.; Vié, V.; Renault, A.; Beaufils, S.; Lefèvre, T.; Paquet-Mercier, F.; Pézolet, M.; Bêche, B.

2013-03-01

327

A new class of animal collagen masquerading as an insect silk  

PubMed Central

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials.

Sutherland, Tara D.; Peng, Yong Y.; Trueman, Holly E.; Weisman, Sarah; Okada, Shoko; Walker, Andrew A.; Sriskantha, Alagacone; White, Jacinta F.; Huson, Mickey G.; Werkmeister, Jerome A.; Glattauer, Veronica; Stoichevska, Violet; Mudie, Stephen T.; Haritos, Victoria S.; Ramshaw, John A. M.

2013-01-01

328

A new class of animal collagen masquerading as an insect silk.  

PubMed

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials. PMID:24091725

Sutherland, Tara D; Peng, Yong Y; Trueman, Holly E; Weisman, Sarah; Okada, Shoko; Walker, Andrew A; Sriskantha, Alagacone; White, Jacinta F; Huson, Mickey G; Werkmeister, Jerome A; Glattauer, Veronica; Stoichevska, Violet; Mudie, Stephen T; Haritos, Victoria S; Ramshaw, John A M

2013-10-04

329

The impact behaviour of silk cocoons.  

PubMed

Silk cocoons, constructed by silkmoths (Lepidoptera), are protective structural composites. Some cocoons appear to have evolved towards structural and material optimisation in order to sustain impact strikes from predators and hinder parasite ingress. This study investigates the protective properties of silk cocoons with different morphologies by evaluating their impact resistance and damage tolerance. Finite element analysis was used to analyse empirical observations of the quasi-static impact response of the silk cocoons, and to evaluate the separate benefits of the structures and materials through the deformation and damage mechanism. We use design principles from composite engineering in order to understand the structure-property-function relationship of silkworm cocoons. Understanding the highly evolved survival strategies of the organisms building natural cocoons will hopefully lead to inspiration that in turn could lead to improved composite design. PMID:23804671

Chen, Fujia; Hesselberg, Thomas; Porter, David; Vollrath, Fritz

2013-07-15

330

Silk fibroin in tissue engineering.  

PubMed

Tissue engineering (TE) is a multidisciplinary field that aims at the in vitro engineering of tissues and organs by integrating science and technology of cells, materials and biochemical factors. Mimicking the natural extracellular matrix is one of the critical and challenging technological barriers, for which scaffold engineering has become a prime focus of research within the field of TE. Amongst the variety of materials tested, silk fibroin (SF) is increasingly being recognized as a promising material for scaffold fabrication. Ease of processing, excellent biocompatibility, remarkable mechanical properties and tailorable degradability of SF has been explored for fabrication of various articles such as films, porous matrices, hydrogels, nonwoven mats, etc., and has been investigated for use in various TE applications, including bone, tendon, ligament, cartilage, skin, liver, trachea, nerve, cornea, eardrum, dental, bladder, etc. The current review extensively covers the progress made in the SF-based in vitro engineering and regeneration of various human tissues and identifies opportunities for further development of this field. PMID:23184771

Kasoju, Naresh; Bora, Utpal

2012-06-04

331

Engineering the Salmonella type III secretion system to export spider silk monomers  

PubMed Central

The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l?1 h?1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology.

Widmaier, Daniel M; Tullman-Ercek, Danielle; Mirsky, Ethan A; Hill, Rena; Govindarajan, Sridhar; Minshull, Jeremy; Voigt, Christopher A

2009-01-01

332

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera.  

PubMed

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C. PMID:21925617

Kameda, Tsunenori; Kojima, Katsura; Zhang, Qiang; Sezutsu, Hideki

2011-09-08

333

Salt-leached silk scaffolds with tunable mechanical properties.  

PubMed

Substrate mechanical properties have remarkable influences on cell behavior and tissue regeneration. Although salt-leached silk scaffolds have been used in tissue engineering, applications in softer tissue regeneration can be encumbered with excessive stiffness. In the present study, silk-bound water interactions were regulated by controlling processing to allow the preparation of salt-leached porous scaffolds with tunable mechanical properties. Increasing silk-bound water interactions resulted in reduced silk II (?-sheet crystal) formation during salt-leaching, which resulted in a modulus decrease in the scaffolds. The microstructures as well as degradation behavior were also changed, implying that this water control and salt-leaching approach can be used to achieve tunable mechanical properties. Considering the utility of silk in various fields of biomedicine, the results point to a new approach to generate silk scaffolds with controllable properties to better mimic soft tissues by combining scaffold preparation methods and silk self-assembly in aqueous solutions. PMID:23016499

Yao, Danyu; Dong, Sen; Lu, Qiang; Hu, Xiao; Kaplan, David L; Zhang, Bingbo; Zhu, Hesun

2012-10-11

334

Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers  

PubMed Central

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline-containing pentaptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX1 GPGGX2)2 with X1/X2=A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX1 GPGGX2)2 with X1/X2 = Y/S or A/A, respectively. Post fiber processing involved similar drawing ratios (2–2.5×) before or after water-treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. NMR indicated that the fraction of ?-sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water-treatment. XRD showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three-dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk-like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif redesign impacted protein self-assembly mechanisms and requirements for fiber formation.

Teule, Florence; Addison, Bennett; Cooper, Alyssa R.; Ayon, Joel; Henning, Robert W.; Benmore, Chris J.; Holland, Gregory P.; Yarger, Jeffery L.; Lewis, Randolph V.

2012-01-01

335

Implication of silk film RGD availability and surface roughness on cytoskeletal organization and proliferation of primary rat bone marrow cells.  

PubMed

To design and fabricate next-generation tissue engineering materials, the understanding of cell responses to material surfaces is required. Surface topography presents powerful cues for cells and can strongly influence cell morphology, adhesion, and proliferation, but the mechanisms mediating this cell response remain unclear. In this report, we have investigated the effects of nanoroughness assemblies of silk fibroin protein membranes and RGD sequences fabricated from two different silk fibroin sources, that is, mulberry (Bombyx mori) and nonmulberry (Antheraea mylitta), on cytoskeletal organization, proliferation, and viability using primary rat bone marrow cells. To vary surface roughness, silk fibroin substrates were treated with graded ethanol (50%-100% v/v) to produce nanoarchitectures in the range of 1-12 nm height. The graded alcohol treatments have been found to produce nanoscale topographies of reproducible height in a much faster and cheaper way. The results showed no difference in cell proliferation within the same treatment groups for both silk types. However, a change in cell response in terms of good cytoskeleton organization, actin development, cell spreading, and strong binding to substratum using A. mylitta fibroin protein films having RGD sequences was observed. This finding provides the information that the nanoroughness affects cellular processes in a cell-specific manner and may be helpful for the development of smart silk-based biomaterials especially for directing cell differentiation and regenerative therapies. PMID:20214452

Mandal, Biman B; Das, Soumen; Choudhury, Koel; Kundu, Subhas C

2010-07-01

336

Preparation of non-woven nanofibers of Bombyx mori silk, Samia cynthia ricini silk and recombinant hybrid silk with electrospinning method  

Microsoft Academic Search

Electrospinning is a good method to obtain nanoscale fibers from polymer solutions. In this paper, we successfully prepared non-woven nanofibers of Bombyx mori and Samia cynthia ricini silk fibroins, and of the recombinant hybrid fiber involving the crystalline domain of B. mori silk and non-crystalline domain of S. c. ricini silk from hexafluoroacetone (HFA) solution using electrospinning method. 13C cross

Kosuke Ohgo; Chenhua Zhao; Mitsuhiro Kobayashi; Tetsuo Asakura

2003-01-01

337

Osteogenic and adipogenic differentiation of rat bone marrow cells on non-mulberry and mulberry silk gland fibroin 3D scaffolds.  

PubMed

This study investigates the potential of 3D silk scaffolds fabricated using tropical tasar non-mulberry, Antheraea mylitta and mulberry, Bombyx mori silk gland fibroin proteins as substrate for osteogenic and adipogenic differentiation of rat bone marrow cells (BMCs). The scaffolds are mechanically robust and show homogenous pore distribution with high porosity and interconnected pore walls. Low immunogenicity of fabricated silk scaffolds as estimated through TNF alpha release indicates its potential as future biopolymeric graft material. Rat bone marrow cells cultured on scaffolds for 28 days under static conditions in osteogenic and adipogenic media respectively led to induction of differentiation. Proliferation and spreading of fibroblasts and bone marrow cells on silk scaffolds were observed to be dependent on scaffold porosity as revealed through confocal microscopic observations. Histological analysis shows osteogenic differentiation within silk scaffolds resulting in extensive mineralization in the form of deposited nodules as observed through intense Alizarin Red S staining. Similarly, adipogenesis was marked by the presence of lipid droplets within scaffolds on staining with Oil Red O. Real-time PCR studies reveal higher transcript levels for osteopontin (Spp1), osteocalcin (Bglap2) and osteonectin (Sparc) genes under osteogenic conditions. Similarly, upregulated adipogenic gene expression was observed within A. mylitta and B. mori scaffolds under adipogenic conditions for Peroxisome proliferator activated receptor gamma (PPARgamma2), lipoprotein lipase (LPL) and adipocyte binding protein (aP2) genes. The results suggest suitability of silk fibroin protein 3D scaffolds as natural biopolymer for potential bone and adipose tissue engineering applications. PMID:19577292

Mandal, Biman B; Kundu, Subhas C

2009-07-03

338

Development of silk-based scaffolds for tissue engineering of bone from human adipose derived stem cells  

PubMed Central

Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and demonstrated ability to support bone formation, in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASC), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol - HFIP), pore sizes (250–500?m vs. 500–1000?m) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes with decellularized trabecular bone (DCB) included as a “gold standard”. The scaffolds were seeded with hASC and cultured for 7 weeks in osteogenic media. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600?m porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2, and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.

Correia, Cristina; Bhumiratana, Sarindr; Yan, Le-Ping; Oliveira, Ana L.; Gimble, Jeffrey M.; Rockwood, Danielle; Kaplan, David L.; Sousa, Rui A.; Reis, Rui L.; Vunjak-Novakovic, Gordana

2012-01-01

339

Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery.  

PubMed

A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs. PMID:22090427

Elia, Roberto; Newhide, Danny R; Pedevillano, Paul D; Reiss, G Russell; Firpo, Matthew A; Hsu, Edward W; Kaplan, David L; Prestwich, Glenn D; Peattie, Robert A

2011-11-15

340

Structure and morphology of regenerated silk nano-fibers produced by electrospinning  

NASA Astrophysics Data System (ADS)

The impressive physical and mechanical properties of natural silk fiberssp1 and the possibility of producing these proteins using biotechnology,sp2 have provided the impetus for recent efforts in both the biosynthesissp{3,4} and the spinning of these protein based biopolymers.sp{5,6,7} The question still remains: whether fibers spun from solutions with similar chemical makeup can produce fibers with similar structures and therefore with the possibility of improved properties. Since genetically engineered silk solutions were not readily available, the first objective of this project was to completely dissolve the Bombyx mori cocoon and the Nephila clavipes dragline silk while maintaining the molecular weight integrity of the polymer. The second objective was to develop a system for re-spinning from very small amount of the resulting silk solutions by the process of electrospinning. The third objective was, to produce regenerated silk fibers with diameters that are several orders of magnitude smaller than the original fibers, suitable for direct observation and analysis by transmission electron microscopy and electron diffraction. And finally, to compare these results to structural information obtained from natural (as spun by the organism) fibers to see if the regenerated solutions are able to form the same structure as the original fibers. Both types of silk fibers were successfully dissolved while maintaining the polymer integrity. Small quantities (25-50 mul) of these solutions were used to electrospin fibers with diameters ranging from 8nm-200nm. The fibers were observed by optical, scanning electron, and transmission electron microscopy. These nano fibers showed optical retardation, appeared to have a circular cross-section, and were dimensionally stable at temperatures above 280sp°C. Electron diffraction patterns of annealed electrospun fibers of B. mori and N. clavipes showed reflections, demonstrating orientational and semicrystalline order in the material comparable to natural silk. In addition, electron diffraction was also obtained form extended microtomed single dragline fibers of N. clavipes, and the d-spacings agreed well with thoes obtained from WAXD of dragline fiber bundles.

Zarkoob, Shahrzad

341

Phononic band gap and mechanical anisotropy in spider silk  

NASA Astrophysics Data System (ADS)

Spider dragline silk is a semi-crystalline biopolymer exhibiting superior properties compared to synthetic polymers with similar chemical structure, such as polyamides. This is ascribed to the hierarchical nanostructure that is created in the spinning duct. During this process the aqueous solution of the two protein constituents of dragline silk is crystallized, while the macromolecules maintain their high orientation, leading to a high value of the Young's modulus (in the order of 10 GPa) along the fiber. We employed spontaneous Brillouin light scattering to measure the longitudinal modulus (M//,,M) along the two symmetry directions of the native fiber with increased (decreased) pre-strain created by stretching (supercontracting after hydration). A strong mechanical anisotropy is found; at about 18% strain M///M˜5. Most important, an unexpected finding is the first observation of a unidirectional hypersonic phononic band gap in biological structures. This relates to the existence of a strain-dependent correlation length of the mechanical modulus in the submicron range along the fiber axis.

Papadopoulos, Periklis; Gomopoulos, Nikos; Kremer, Friedrich; Fytas, George

2010-03-01

342

Structure and properties of silk grafted with acrylate fluoride monomers by ATRP  

NASA Astrophysics Data System (ADS)

In order to develop water repellence silk materials, silk was grafted using acrylate fluoride monomers via atom transfer radical polymerization (ATRP) method. Scanning electron microscopy (SEM) photos of the grafted silks showed significant difference from the untreated silk. FT-IR characterization of the modified silk substrate indicated that acrylate fluoride monomers were successfully grafted onto silk surface. Differential scanning calorimetry (DSC) curves indicated that the thermal stability of the grafted silk was improved. The whiteness, breaking strength, elongation at break and air permeability of the grafted silk fabrics decreased slightly compared with the control sample. Surface contact angle test and water repellency rating test showed that the water repellence of the modified silk fabrics were better than the untreated silk. Functional silk fabric with good water repellence property could be obtained by properly controlling the grafting yield, which had little effect on the intrinsic properties of silk fabric.

Li, Shiwei; Xing, Tieling; Li, Zhanxiong; Chen, Guoqiang

2013-03-01

343

The use of injectable sonication-induced silk hydrogel for VEGF165 and BMP-2 delivery for elevation of the maxillary sinus floor  

PubMed Central

Sonication-induced silk hydrogels were previously prepared as an injectable bone replacement biomaterial, with a need to improve osteogenic features. Vascular endothelial growth factor (VEGF165) and bone morphogenic protein-2 (BMP-2) are key regulators of angiogenesis and osteogenesis, respectively, during bone regeneration. Therefore, the present study aimed at evaluating in situ forming silk hydrogels as a vehicle to encapsulate dual factors for rabbit maxillary sinus floor augmentation. Sonication-induced silk hydrogels were prepared in vitro and the slow release of VEGF165 and BMP-2 from these silk gels was evaluated by ELISA. For in vivo studies for each time point (4 and 12 weeks), 24 sinus floors elevation surgeries were made bilaterally in 12 rabbits for the following four treatment groups: silk gel (group Silk gel), silk gel/VEGF165 (group VEGF), silk gel/BMP-2 (group BMP-2), silk gel/VEGF165/BMP-2 (group V+B) (n=6 per group). Sequential florescent labeling and radiographic observations were used to record new bone formation and mineralization, along with histological and histomorphometric analysis. At week 4, VEGF165 promoted more tissue infiltration into the gel and accelerated the degradation of the gel material. At this time point, the bone area in group V+B was significantly larger than those in the other three groups. At week 12, elevated sinus floor heights of groups BMP-2 and V+B were larger than those of the Silk gel and VEGF groups, and the V+B group had the largest new bone area among all groups. In addition, a larger blood vessel area formed in the remaining gel areas in groups VEGF and V+B. In conclusion, VEGF165 and BMP-2 released from injectable and biodegradable silk gels promoted angiogenesis and new bone formation, with the two factors demonstrating an additive effect on bone regeneration. These results indicate that silk hydrogels can be used as an injectable vehicle to deliver multiple growth factors in a minimally invasive approach to regenerate irregular bony cavities.

Zhang, Wenjie; Wang, Xiuli; Wang, Shaoyi; Zhao, Jun; Xu, Lianyi; Zhu, Chao; Zeng, Deliang; Chen, Jake; Zhang, Zhiyuan; Kaplan, David L.; Jiang, Xinquan

2011-01-01

344

Silk ionomers for encapsulation and differentiation of human MSCs.  

PubMed

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages. PMID:22824008

Calabrese, Rossella; Kaplan, David L

2012-07-21

345

Silk ionomers for encapsulation and differentiation of human MSCs  

PubMed Central

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.

Calabrese, Rossella; Kaplan, David L.

2012-01-01

346

The Ancient Art of Silk Painting  

ERIC Educational Resources Information Center

|In this article, the author describes a silk-painting project with a sea-creature theme for eighth-grade students. Other themes can be used such as geometric quilt designs, tropical rain forest, large flowers, Art Nouveau motifs, portraits and more. (Contains 2 resources.)|

Yonker, Kim

2010-01-01

347

Constructing Knowledge with Silk Road Visuals  

ERIC Educational Resources Information Center

|In this study a group of elementary teachers use illustrations, rather than written text, to introduce their students to the peoples and places of the ancient silk routes. The illustrations are from two picture books; "Marco Polo," written by Gian Paolo Cesaerani and illustrated by Piero Ventura (1977), and "We're Riding on a Caravan: An…

Bisland, Beverly Milner

2008-01-01

348

Multiple recombining loci encode MaSp1, the primary constituent of dragline silk, in widow spiders (Latrodectus: Theridiidae).  

PubMed

Spiders spin a functionally diverse array of silk fibers, each composed of one or more unique proteins. Most of these proteins, in turn, are encoded by members of a single gene family thought to have arisen through duplication and divergence of an ancestral silk gene. Because of its remarkable mechanical properties, orb weaver dragline silk, a composite of 2 proteins (MaSp1 and MaSp2), is the best studied. Here, we demonstrate that multiple loci encode MaSp1 in widow spiders (Latrodectus). Because these copies may be the result of more recent duplication events than those leading to the currently recognized silk gene paralogs, they offer insight into the early evolutionary fate of silk gene duplicates. In addition to 3 presumed functional MaSp1 loci in Latrodectus hesperus (Western black widow) and Latrodectus geometricus (brown widow) genomes, we find a MaSp1 pseudogene in L. hesperus, demonstrating the potential for unrecognized extinction of silk gene paralogs. We also document recombination events among L. hesperus MaSp1 loci and between Latrodectus MaSp1 loci and MaSp2. This result supports the hypothesis that concerted evolution occurs not only within an individual silk gene but also among silk gene paralogs. One of the L. geometricus MaSp1 copies encodes a protein that has diverged in amino acid composition and potentially converged on the secondary structure of MaSp2. Based on the presence of multiple MaSp1 loci and the phylogenetic distribution of MaSp1 versus MaSp2, we propose that MaSp2 is derived from an ancestral MaSp1 duplicate. Finally, divergence has occurred in the upstream flanking sequences of the L. hesperus MaSp1 loci, the region most likely to contain regulatory motifs, providing ample opportunity for differential expression. However, the benefits associated with increased protein production may be the primary mechanism maintaining multiple functional MaSp1 copies in widow genomes. PMID:18048404

Ayoub, Nadia A; Hayashi, Cheryl Y

2007-11-28

349

The Influence of Elasticity and Surface Roughness on Myogenic and Osteogenic-Differentiation of Cells on Silk-Elastin Biomaterials  

PubMed Central

The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.

Hu, Xiao; Park, Sang-Hyug; Gil, Eun Seok; Xia, Xiao-Xia; Weiss, Anthony S.; Kaplan, David L.

2011-01-01

350

The Preparation and Characterization of Silk\\/Gelatin Biocomposites  

Microsoft Academic Search

There is a growing interest in the use of composite materials. Silk fiber\\/gelatin biocomposites were fabricated using compression molding. The fiber content in the composite varied from 10–30 wt%. Composite containing 30 wt% silk showed the best mechanical properties. Tensile strength, tensile modulus, bending strength, bending modulus and impact strength, hardness of the 30% silk content composites were found 54 MPa, 0.95 GPa, 75 MPa

Quazi T. H. Shubhra; A. K. M. M. Alam; M. A. Khan; M. Saha; Dipti Saha; Jahangir A. Khan; M. A. Quaiyyum

2010-01-01

351

Silk production in a spider involves acid bath treatment  

PubMed Central

We studied physiological conditions during the spinning of dragline silk by the garden cross spider, Araneus diadematus. Silk is converted from the liquid feedstock in the gland into a solid thread via a tapering tubular duct and exits at a spigot. The distal part of the tubule appears specialized for ion transport and the management of the pH inside the lumen. Thus, it appears that spider silk in vivo, like some industrial polymers in vitro, is spun through an acid bath.

Vollrath, F.; Knight, D. P.; Hu, X. W.

1998-01-01

352

Controlling the molecular structure and physical properties of artificial honeybee silk by heating or by immersion in solvents.  

PubMed

Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of ß-sheets, with the amount of ß-sheet dictated by methanol concentration. Formation of ß-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190°C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties. PMID:23300639

Huson, Mickey G; Church, Jeffrey S; Poole, Jacinta M; Weisman, Sarah; Sriskantha, Alagacone; Warden, Andrew C; Campbell, Peter M; Ramshaw, John A M; Sutherland, Tara D

2012-12-21

353

Low-threshold blue lasing from silk fibroin thin films  

NASA Astrophysics Data System (ADS)

Silk is a natural biocompatible material that can be integrated in a variety of photonic systems and optoelectronic devices. The silk replication of patterned substrates with features down to tens of nanometers is exploited to realize highly transparent, mechanically stable, and free-standing structures with optical wavelength size. We demonstrate organic lasing from a blue-emitting stilbene-doped silk film spin-coated onto a one-dimensional distributed feedback grating (DFB). The lasing threshold is lower than that of organic DFB lasers based on the same active dye. These findings pave the way to the development of an optically active biocompatible technological platform based on silk.

Toffanin, Stefano; Kim, Sunghwan; Cavallini, Susanna; Natali, Marco; Benfenati, Valentina; Amsden, Jason J.; Kaplan, David L.; Zamboni, Roberto; Muccini, Michele; Omenetto, Fiorenzo G.

2012-08-01

354

Regenerated cellulose-silk fibroin blends fibers.  

PubMed

Fibers made of cellulose and silk fibroin at different composition were wet spun from solutions by using N-methylmorpholine N-oxide hydrates (NMMO/H(2)O) as solvent and ethanol as coagulant. Different spinning conditions were used. The fibers were characterized by different techniques: FTIR-Raman, scanning electron microscopy, wide-angle x-ray diffraction, DSC analysis. The results evidence a phase separation in the whole blends compositions. The tensile characterization, however, illustrates that the properties of the blends fibers are higher respect to a linear behaviour between the pure polymers, confirming a good compatibility between cellulose and silk fibroin. The fibers containing 75% of cellulose show better mechanical properties than pure cellulose fibers: modulus of about 23 GPa and strength to break of 307 MPa. PMID:18513793

Marsano, Enrico; Corsini, Paola; Canetti, Maurizio; Freddi, Giuliano

2008-04-11

355

Unique molecular architecture of silk fibroin in the waxmoth, Galleria mellonella.  

PubMed

Proteins of silk fibers are characterized by reiterations of amino acid repeats. Physical properties of the fiber are determined by the amino acid composition, the complexity of repetitive units, and arrangement of these units into higher order arrays. Except for very short motifs of 6-10 residues, the length of repetitive units and the number of these units concatenated in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes an exceptional silk protein represented by the 500-kDa heavy chain fibroin (H-fibroin) of the waxmoth, Galleria mellonella. Its non-repetitive N-terminal (175 residues) and C-terminal (60 residues) parts, the overall gene organization, and the nucleotide sequence around the TATA box show that it is homologous to the H-fibroins of other Lepidoptera. However, over 95% of the protein consists of highly ordered repetitive structures that are unmatched in other species. The repetitive region includes 11 assemblies AB(1)AB(1)AB(1)AB(2)(AB(2))AB(2) of remarkably conserved polypeptide repeats A (63 amino acid residues), B(1) (43 residues), and B(2) (18 residues). The repeats contain a high proportion of Gly (31.6%), Ala (23.8%), Ser (18.1%), and of residues with long hydrophobic side chains (16% for Leu, Ile, and Val combined). The presence of the GLGGLG and SSAASAA(AA) motifs suggests formation of pleated beta-sheets and their stacking into crystallites. Conspicuous conservation of the apolar sequence VIVI followed by DD or ED is interpreted as indicating the importance of hydrophobicity and electrostatic charge in H-fibroin cross-linking. The environment of G. mellonella larvae within bee cultures requires continuous production of silk that must be both strong and elastic. The spectacular arrangement of the repetitive H-fibroin region apparently evolved to meet these requirements. PMID:11886872

Zurovec, Michal; Sehnal, Frantisek

2002-03-08

356

A proposed model for dragline spider silk self-assembly: insights from the effect of the repetitive domain size on fiber properties.  

PubMed

Dragline spider silk has been intensively studied for its superior qualities as a biomaterial. In previous studies, we made use of the baculovirus mediated expression system for the production of a recombinant Araneus diadematus spider silk dragline ADF4 protein and its self-assembly into intricate fibers in host insect cells. In this study, our aim was to explore the function of the major repetitive domain of the dragline spider silk. Thus, we generated an array of synthetic proteins, each containing a different number of identical repeats up to the largest recombinantly expressed spider silk to date. Study of the self-assembly properties of these proteins showed that depending on the increasing number of repeats they give rise to different assembly phenotypes, from a fully soluble protein to bona fide fibers with superior qualities. The different assembly forms, the corresponding chemical resistance properties obtained as well as ultrastructural studies, revealed novel insights concerning the structure and intermolecular interactions of the repetitive and nonrepetitive domains. Based on these observations and current knowledge in the field, we hereby present a comprehensive hypothetical model for the mechanism of dragline silk self-assembly and fiber formation. PMID:20014164

Ittah, Shmulik; Barak, Noaa; Gat, Uri

2010-05-01

357

Directional water collection on wetted spider silk.  

PubMed

Many biological surfaces in both the plant and animal kingdom possess unusual structural features at the micro- and nanometre-scale that control their interaction with water and hence wettability. An intriguing example is provided by desert beetles, which use micrometre-sized patterns of hydrophobic and hydrophilic regions on their backs to capture water from humid air. As anyone who has admired spider webs adorned with dew drops will appreciate, spider silk is also capable of efficiently collecting water from air. Here we show that the water-collecting ability of the capture silk of the cribellate spider Uloborus walckenaerius is the result of a unique fibre structure that forms after wetting, with the 'wet-rebuilt' fibres characterized by periodic spindle-knots made of random nanofibrils and separated by joints made of aligned nanofibrils. These structural features result in a surface energy gradient between the spindle-knots and the joints and also in a difference in Laplace pressure, with both factors acting together to achieve continuous condensation and directional collection of water drops around spindle-knots. Submillimetre-sized liquid drops have been driven by surface energy gradients or a difference in Laplace pressure, but until now neither force on its own has been used to overcome the larger hysteresis effects that make the movement of micrometre-sized drops more difficult. By tapping into both driving forces, spider silk achieves this task. Inspired by this finding, we designed artificial fibres that mimic the structural features of silk and exhibit its directional water-collecting ability. PMID:20130646

Zheng, Yongmei; Bai, Hao; Huang, Zhongbing; Tian, Xuelin; Nie, Fu-Qiang; Zhao, Yong; Zhai, Jin; Jiang, Lei

2010-02-01

358

The Silk Road in World History  

Microsoft Academic Search

The Silk Road was the current name for a complex of ancient trade routes linking East Asia with Central Asia, South Asia, and the Mediterranean world. This network of exchange emerged along the borders between agricultural China and the steppe nomads during the Han Dynasty (206BCE-220CE), in consequence of the inter-dependence and the conflicts of these two distinctive societies. In

Xinru Liu

359

In vitro degradation of silk fibroin  

Microsoft Academic Search

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1mg\\/ml Protease XIV at 37°C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1)

Rebecca L. Horan; Kathryn Antle; Adam L. Collette; Yongzhong Wang; Jia Huang; Jodie E. Moreau; Vladimir Volloch; David L. Kaplan; Gregory H. Altman

2005-01-01

360

Biodegradable materials based on silk fibroin and keratin.  

PubMed

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles. PMID:18355027

Vasconcelos, Andreia; Freddi, Giuliano; Cavaco-Paulo, Artur

2008-03-21

361

Templated Native Silk Smectic Gels.  

National Technical Information Service (NTIS)

One aspect of the present invention relates to a method of preparing a fibrous protein smectic hydrogel by way of a solvent templating process, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that ...

H. H. Jin J. H. Park R. Valluzzi

2003-01-01

362

Anti-inflammation effects of corn silk in a rat model of carrageenin-induced pleurisy.  

PubMed

Pleurisy is an inflammation of the pleural layers that surround the lungs. Despite much research into inflammatory diseases, no drugs with favorable safety profiles are available yet for their treatment. Corn silk has been used in many parts of the world for the treatment of edema, cystitis, gout, kidney stones nephritis, and prostitutes. However, no scientific reports on the anti-inflammatory effects of corn silk were so far available. To test the anti-inflammatory efficacy of corn silk extract (CSEX) in a rat model of carrageenin (Cg)-induced pleurisy, exudate formation, and cellular infiltration, tumor necrosis factor alpha (TNF-?), interleukin 1 beta (IL-1?), vascular endothelial growth factor alpha (VEGF-?), interleukin-17 (IL-17), C3 and C4 complement protein levels, adhesion molecule (ICAM-1) and inducible nitric oxide synthase (iNOS) levels, nuclear factor kappa B (NF-?B) activation, and total antioxidant activity were studied, respectively. Pretreatment with CSEX reduced Cg-induced pleurisy exudate, number of leukocytes, oxidative stress, C3 protein level, and O (2)(-) levels at the inflammatory site. Pretreatment with CSEX also inhibited TNF-?, IL-1?, VEGF-?, and IL-17A and blocked inflammation-related events (ICAM-1 and iNOS) by activation of NF-?B. Supplementation with CSEX may be a promising treatment for inflammatory diseases that involve oxidative stress. PMID:21898269

Wang, Guang-Qiang; Xu, Tao; Bu, Xue-Mei; Liu, Bao-Yi

2012-06-01

363

Cell cycle events during the development of the silk glands in the mulberry silkworm Bombyx mori  

Microsoft Academic Search

Silk glands of the mulberry silkworm Bombyx mori are long and paired structures originating from the labial region and are anatomically and physiologically divided into three major compartments, the anterior, middle and posterior silk glands. The silk gland morphogenesis is complete by 8 days post egg laying. Extensive growth of silk glands during the larval stages is due to increase in

Sangeeta Dhawan; K. P. Gopinathan

2003-01-01

364

Innovative and Multidirectional Applications of Natural Fibre, Silk - A Review  

Microsoft Academic Search

Realization of current trends and innovative uses of insect fibre, silk is required to exploit its compatibility, eco friendly and value addition potential. The nutritive value as human diet for cardiac and diabetic patients, component for cosmetic preparations indicate silk application rate. The bio-compatibility made it a base material for tissue wall, membrane, muscle ligament, blood vessel, nerve gadget, cartilage

R. Manohar Reddy

365

First investigation of spider silk as a braided microsurgical suture.  

PubMed

Inhibition of axonal outgrowth accompanied by neuroma formation appears in microsurgical nerve repair as reaction to common microsuture materials like silk, nylon, or polyglycolic acid. In contrast, recent findings revealed advantages of spider silk fibers in guiding Schwann cells in nerve regeneration. Here, we asked if we could braid microsutures from native spider silk fibers. Microsutures braided of native spider dragline silk were manufactured, containing either 2 × 15 or 3 × 10 single fibres strands. Morphologic appearance was studied and tensile strength and stress-strain ratio (SSR) were calculated. The constructed spider silk sutures showed a median thickness of 25 ?m, matching the USP definition of 10-0. Maximum load and tensile strength for both spider silk microsutures were significantly more than 2-fold higher than for nylon suture; SSR was 1.5-fold higher. All values except elasticity were higher in 3 × 10 strand sutures compared to 2 × 15 strand sutures, but not significantly. In this pilot study, we demonstrate the successful manufacture of microsutures from spider silk. With regards to the mechanical properties, these sutures were superior to nylon sutures. As spider silk displays high biocompatibility in nerve regeneration, its usage in microsurgical nerve repair should be considered. PMID:21432995

Kuhbier, Joern W; Reimers, Kerstin; Kasper, Cornelia; Allmeling, Christina; Hillmer, Anja; Menger, Björn; Vogt, Peter M; Radtke, Christine

2011-03-22

366

Animal silks: their structures, properties and artificial production.  

PubMed

This feature article reviews recent progress in the understanding of the hierarchically organized structures, the perfectly balanced mechanical properties and the structure-property relationship of the natural animal silk fibres, as well as the experimental attempts to fabricate man-made silk fibres by means of wet spinning, dry spinning, electrospinning and transgenosis. PMID:19865641

Fu, Chengjie; Shao, Zhengzhong; Fritz, Vollrath

2009-09-08

367

Coupling time to silking with plant growth rate in maize  

Microsoft Academic Search

In maize (Zea mays L.), progress towards pistillate flower maturity (silking) is highly dependent upon the environmental conditions around flowering. Under conditions that inhibit plant growth, female flower development is delayed relative to that of the male flowers resulting in an increase in the anthesis–silking interval (ASI). Although variation in ASI has been extensively documented, its relationship to plant growth

Lucas Borrás; Mark E. Westgate; Juan P. Astini; Laura Echarte

2007-01-01

368

Geographic Perspectives with Elementary Students: The Silk Road  

ERIC Educational Resources Information Center

|The purpose of this study is to investigate elementary students' explanations of how physical features of the land influence the location of humanly defined structures including trade routes, such as the silk routes. The silk routes were a series of caravan trade routes that extended from Turkey to China and were located as far south as India and…

Bisland, Beverly Milner

2006-01-01

369

Visual Literacy with Picture Books: The Silk Road  

ERIC Educational Resources Information Center

|The ancient Silk Routes connecting China to Europe across the rugged mountains and deserts of central Asia are one of the primary examples of transculturation in world history. Traders on these routes dealt not only in goods such as silk and horses but also made possible the spread of art forms as well as two major religions, Buddhism and Islam.…

Bisland, Beverly Milner Lee

2007-01-01

370

Project for a Comprehensive Study of the Silk Roads  

Microsoft Academic Search

From time immemorial the Silk Road, with its hundreds of byways, was considered the highway that linked the worlds of East and West. In the mid dle of the twentieth century, scholarly research revealed the crucial role played by oases in linking the North to the South; these oases, even more than the transversal lines of the Silk Roads, played

Vadim Eliseyev

1995-01-01

371

Synthesis of silver chloride nanocrystal on silk fibers  

Microsoft Academic Search

The synthesis of silver chloride nanocrystals on silk fiber is reported. The growth of the nanocrystal was achieved by sequential dipping of the silk fibers in alternating solution of either silver nitrate or sodium chloride followed by a rinse step. The negative charge present at the surface of the fibers can immobilize silver ions which react in the next sodium

Pranut Potiyaraj; Panittamat Kumlangdudsana; Stephan T. Dubas

2007-01-01

372

Intervertebral Disk Tissue Engineering Using Biphasic Silk Composite Scaffolds  

PubMed Central

Scaffolds composed of synthetic, natural, and hybrid materials have been investigated as options to restore intervertebral disk (IVD) tissue function. These systems fall short of the lamellar features of the native annulus fibrosus (AF) tissue or focus only on the nucleus pulposus (NP) tissue. However, successful regeneration of the entire IVD requires a combination approach to restore functions of both the AF and NP. To address this need, a biphasic biomaterial structure was generated by using silk protein for the AF and fibrin/hyaluronic acid (HA) gels for the NP. Two cell types, porcine AF cells and chondrocytes, were utilized. For the AF tissue, two types of scaffold morphologies, lamellar and porous, were studied with the porous system serving as a control. Toroidal scaffolds formed out of the lamellar, and porous silk materials were used to generate structures with an outer diameter of 8?mm, inner diameter of 3.5?mm, and a height of 3?mm (the interlamellar distance in the lamellar scaffold was 150–250??m, and the average pore sizes in the porous scaffolds were 100–250??m). The scaffolds were seeded with porcine AF cells to form AF tissue, whereas porcine chondrocytes were encapsulated in fibrin/HA hydrogels for the NP tissue and embedded in the center of the toroidal disk. Histology, biochemical assays, and gene expression indicated that the lamellar scaffolds supported AF-like tissue over 2 weeks. Porcine chondrocytes formed the NP phenotype within the hydrogel after 4 weeks of culture with the AF tissue that had been previously cultured for 2 weeks, for a total of 6 weeks of cultivation. This biphasic scaffold simulating in combination of both AF and NP tissues was effective in the formation of the total IVD in vitro.

Park, Sang-Hyug; Gil, Eun Seok; Cho, Hongsik; Mandal, Biman B.; Tien, Lee W.; Min, Byoung-Hyun

2012-01-01

373

P25 gene regulation in Bombyx mori silk gland: two promoter-binding factors have distinct tissue and developmental specificities.  

PubMed Central

The gene encoding the silk protein P25 is expressed in the posterior silk gland of Bombyx mori with strict territorial and developmental specificities. The cis-acting regulatory elements previously located within the 441-bp 5' proximal sequence of the gene were examined for protein-binding capacities. We identified two factors, BMFA and SGFB, that lead to prominent band shifts and the target sites for which are included in a region homologous to the fibroin gene enhancer sequence. Analysis of the tissue-specific incidence of both factors showed that BMFA is ubiquitous, whereas SGFB is restricted to the silk gland cells. However, SGFB was found in both posterior and middle silk gland cells and therefore likely directs organ-specific, but not territory-specific, expression. Developmental studies throughout the fourth larval molt, at which the P25 gene status changes from derepressed to repressed, revealed that BMFA is reversibly modified at the transition from intermolt to molt. Indeed, the preexisting BMFA is replaced by a structurally related factor, BMFA', during the 2 h following head capsule apolysis. The exact temporal coincidence of this conversion with the onset of gene repression suggests that BMFA' is involved in transcription inactivation and likely results from a transduction process initiated by the hormonal change at molting. Images

Durand, B; Drevet, J; Couble, P

1992-01-01

374

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin, gelatin and hydroxyapatite  

Microsoft Academic Search

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin\\/Thai silk fibroin (CGSF), hydroxyapatite\\/Thai\\u000a silk fibroin (SF4), and hydroxyapatite\\/conjugated gelatin\\/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen\\u000a leaching, dehydrothermal\\/chemical crosslinking and an alternate soaking technique for mineralization. In

Hathairat Tungtasana; Somruetai Shuangshoti; Shanop Shuangshoti; Sorada Kanokpanont; David L. Kaplan; Tanom Bunaprasert; Siriporn Damrongsakkul

2010-01-01

375

Multifunctionalized electrospun silk fibers promote axon regeneration in central nervous system  

PubMed Central

The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.

Wittmer, Corinne R.; Claudepierre, Thomas; Reber, Michael; Wiedemann, Peter; Garlick, Jonathan A.; Kaplan, David

2012-01-01

376

Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering  

PubMed Central

RGD-coupled silk protein-biomaterial lamellar systems were prepared and studied with human cornea fibroblasts (hCFs) to match functional requirements. A strategy for corneal tissue engineering was pursued to replicate the structural hierarchy of human corneal stroma within thin stacks of lamellae-like tissues, in this case constructed from scaffolds constructed with RGD-coupled, patterned, porous, mechanically robust and transparent silk films. The influence of RGD-coupling on the orientation, proliferation, ECM organization, and gene expression of hCFs was assessed. RGD surface modification enhanced cell attachment, proliferation, alignment and expression of both collagens (type I and V) and proteoglycans (decorin and biglycan). Confocal and histological images of the lamellar systems revealed that the bio-functionalized silk human cornea 3D constructs exhibited integrated corneal stroma tissue with helicoidal multi-lamellar alignment of collagen-rich and proteoglycan-rich extracellular matrix, with transparency of the construct. This biomimetic approach to replicate corneal stromal tissue structural hierarchy and architecture demonstrates a useful strategy for engineering human cornea. Further, this approach can be exploited for other tissue systems due to the pervasive nature of such helicoids in most human tissues.

Gil, Eun Seok; Mandal, Biman B.; Park, Sang-Hyug; Marchant, Jeffrey K.; Omenetto, Fiorenzo G.; Kaplan, David L.

2010-01-01

377

Diversity of molecular transformations involved in the formation of spider silks.  

PubMed

Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Taking advantage of the efficiency of Raman spectromicroscopy in investigating micrometer-sized biological samples, we have determined the secondary structure of proteins in the complete set of glands of the orb-weaving spider Nephila clavipes. The major and minor ampullate silks in the sac of their glands have identical secondary structures typical of natively unfolded proteins. Spidroins are converted into fibers containing highly oriented ?-sheets. The capture spiral represents a distinct structural singleton. The proteins are highly disordered prior to spinning and undergo no molecular change or alignment upon spinning. The cylindrical, aciniform, and piriform proteins are folded in their initial state with a predominance of ?-helices, but whereas the cylindrical gland forms a fiber similar to the major ampullate thread, the aciniform and piriform glands produce fibers dominated by moderately oriented ?-sheets and ?-helices. The conformation of the proteins before spinning is related to intrinsic characteristics of their primary structure. Proteins that are unfolded in the gland have repeat sequences composed of submotifs and display no sequence regions with aggregation propensity. By contrast, the folded proteins have neither submotifs nor aggregation-prone sequence regions. Taken together, the Raman data show a remarkable diversity of molecular transformations occurring upon spinning. PMID:21050860

Lefèvre, Thierry; Boudreault, Simon; Cloutier, Conrad; Pézolet, Michel

2010-11-02

378

Modular evolution of egg case silk genes across orb-weaving spider superfamilies.  

PubMed

Spider silk proteins (fibroins) are renowned for their extraordinary mechanical properties and biomimetic potential. Despite extensive evolutionary, ecological, and industrial interest in these fibroins, only a fraction of the known silk types have been characterized at the molecular level. Here we report cDNA and genomic sequences of the fibroin TuSp1, which appears to be the major component of tubuliform gland silk, a fiber exclusively synthesized by female spiders for egg case construction. We obtained TuSp1 sequences from 12 spider species that represent the extremes of phylogenetic diversity within the Orbicularia (orb-weaver superfamilies, Araneoidea and Deinopoidea) and finer scale sampling within genera. TuSp1 encodes tandem arrays of an approximately 200-aa-long repeat unit and individual repeats are readily aligned, even among species that diverged >125 million years ago. Analyses of these repeats across species reveal the strong influence of concerted evolution, resulting in intragenic homogenization. However, deinopoid TuSp1 repeats also contain insertions of coding, minisatellite-like sequences, an apparent result of replication slippage and nonreciprocal recombination. Phylogenetic analyses of 37 spider fibroin sequences support the monophyly of TuSp1 within the spider fibroin gene family, consistent with a single origin of this ortholog group. The diversity of taxa and silks examined here confirms that repetitive architecture is a general feature of this gene family. Moreover, we show that TuSp1 provides a clear example of modular evolution across a range of phylogenetic levels. PMID:16061817

Garb, Jessica E; Hayashi, Cheryl Y

2005-08-01

379

Effect of pineapple protease on the characteristics of protein fibers  

Microsoft Academic Search

A pineapple protease, bromelain, was used to improve the dyeing properties of protein fibers such as wool and silk. The optimal\\u000a condition for the activity of the pineapple protease was about 60 °C at pH 7. The wool and silk were treated with the protease\\u000a extracted from a pineapple and the K\\/S values of the dyed wool and silk were

Joonseok Koh; Sang-Mo Kang; Soo-Jin Kim; Min-Kyung Cha; Yoon-Jung Kwon

2006-01-01

380

Biomimetic calcium phosphate coatings on recombinant spider silk fibres.  

PubMed

Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant minispidroins. In the present study, supersaturated simulated body fluid was used to deposit calcium phosphate coatings on recombinant spider silk fibres. The mineralization process was followed in time using scanning electron microscopy equipped with an energy dispersive x-ray (EDX) detector and Raman spectroscope. Focused ion beam technology was used to produce a cross section of a coated fibre, which was further analysed by EDX. Preliminary in vitro experiments using a culture of bone marrow-derived human mesenchymal stem cells (hMSCs) on coated fibres were also performed. This study showed that recombinant spider silk fibres were successfully coated with a homogeneous and thick crystalline calcium phosphate layer. In the course of the mineralization process from modified simulated body fluid, sodium chloride crystals were first deposited on the silk surface, followed by the deposition of a calcium phosphate layer. The coated silk fibres supported the attachment and growth of hMSCs. PMID:20539057

Yang, Liang; Hedhammar, My; Blom, Tobias; Leifer, Klaus; Johansson, Jan; Habibovic, Pamela; van Blitterswijk, Clemens A

2010-06-11