In situ detection of silk fibroin using a dual recognition strategy with a flexible pressure immunosensor.

Silk is a symbol of ancient Chinese civilization that has made an indelible contribution to the development of world civilization. However, because ancient artifacts are often contaminated or degraded, it is difficult to detect the presence of silk therein, and the true origin of silk thus remains a mystery. Therefore, this work presents a flexible pressure immunosensor that was designed based on 3D polypyrrole (PPy) foams for the trace detection of silk fibroin at archaeological sites. Initially, silk fibroin (SF) was conjugated with antibody-functionalized copper oxide nanoparticles (CuO NPs) and carboxylated magnetic beads (MBs) to form a sandwich immune complex. Then, the sandwich immune complex was added to hydrogen peroxide (H2O2) by magnetic separation to catalyse the generation of oxygen (O2), which converted the antigen-antibody specific recognition signal to gas pressure. As the pressure within the device increases, the 3D PPy foam, as the sensing layer resistance was 150 Ω, undergoes extrusion and deformation. This deformation leads to alterations in the foam resistance. The flexible pressure immunosensor can sensitively monitor the change in electrical resistance in the system and quantitatively detect silk fibroin. With optimization, the flexible pressure immunosensor demonstrates a dynamic range of operation spanning from 10 ng mL-1 to 100 µg mL-1, exhibiting a remarkable detection limit of 10.58 ng mL-1 specifically for silk fibroin. Notably, this immunosensor surpasses enzyme-linked immunosorbent assay (ELISA) in terms of superior reproducibility, specificity, and accuracy. Therefore, this application provides a new method and technical support for silk detection.

Microbial degradation mechanism of historical silk revealed by proteomics and metabolomics.

Archaeological silk undergoes destructive and irreversible changes during the natural process of decay. However, in-depth studies on the influence of this biological factor are still lacking. Here, a combination of proteomics and metabolomics is proposed for the first time to explore the interaction between bacteria and historical silk during biodegradation, which provides information on changes at the molecular level of proteins and bacterial metabolites. Morphological observation revealed biofilms produced by Stenotrophomonas maltophilia and Pseudomonas alcaligenes when cultured in the stationary phase and confirmed severe deterioration of silk. Proteomics showed that S. maltophilia had an unbiased effect on silk fibroin, indicating its ability to disrupt both heavy and light chains, as well as other proteins, while P. alcaligenes showed an affinity for more disordered proteins. Analysis of bacterial metabolites showed that overall activity reduction and significant accumulation of fatty acid and phenol metabolites occurred after silk addition, suggesting that the presence of silk may inhibit the activity of an individual strain. This study provides a new insight into the microbial degradation mechanism of archaeological silk.

Proteomics and Immunology Provide Insight into the Degradation Mechanism of Historic and Artificially Aged Silk.

The process and mechanism of silk degradation is still a bewildering mystery in the investigation and conservation of cultural relics, which rely on the development of accurate and tailored analysis technologies. Here, two advanced approaches, proteomics and immunology, were developed for determining the deterioration behavior of historic silk fabrics and artificially aged samples from the molecular to the holistic level. The surface morphology and secondary structure of silk were destroyed during degradation. Subsequently, the proteomics and immunology analysis demonstrated a new degradation model differing from previous reports. First, the amorphous region and the looser crystalline regions were destroyed together, and the macromolecular chains were broken randomly. Then, the tight ß-sheet blocks in the crystalline region were exposed and deteriorated, which expedited the degradation of tight ß-sheet blocks and relatively loose blocks in the crystalline domain as well as the amorphous domain, ultimately yielding small molecule polypeptides. However, the deterioration process of ancient fabrics could be accelerated by poor burial conditions, thus showing distinct destructive characteristics. Overall, the results gave us a more comprehensive and profound understanding of the degradation process of ancient silk.

Ultrasensitive Electrochemical Immunosensor Reveals the Existence of Silk Products on the Maritime Silk Road.

The Maritime Silk Road was the major trade route between eastern and western civilizations in the Middle Ages. However, hardly any silk products have been found along the transoceanic trade route. Thus, the extrasensitive detection of silk relic traces has tremendous importance in research regarding the Maritime Silk Road. In this study, an electrochemical immunosensor based on a tailored monoclonal antibody and gold nanoparticles using the layer-by-layer self-assembly method was devised. The fabricated immunosensor demonstrated preeminent performance in the analysis of silk fibroin, with a linear detection range of 0.01-100 ng mL-1 and a detection limit of 3.8 pg mL-1. In particular, the performance of the immunosensor was excellent in the analysis of ancient silk samples, especially in the qualitative and quantitative detection of soil samples extracted from Nanhai No. 1 shipwreck archeological sites. The proposed electrochemical immunosensor proves the existence of silk products on the Maritime Silk Road and demonstrates enormous potential for studying the formation and development of the ancient transoceanic trading route.

Monitoring of indoor polycyclic aromatic hydrocarbons using spider webs.

Fifteen polycyclic aromatic hydrocarbons (PAHs) were measured in the spider webs prepared in the laboratory and exposed to indoor air pollution in a defined period of time. We have selected homes differing in location (rural area vs. city), type of room (living room, kitchen, basement), inhabitants' habits (smoking cigarettes vs. non-smoking) and type of heating/cooking devices used (natural gas, liquefied gas, coal- and wood-fuelled heating). Webs from two species, from Agelenidae and Pholcidae families, were prepared and used for monitoring of PAHs. PAHs were characterised based on concentration, profile distribution, source apportionment by cluster analysis and diagnostic ratios. The concentrations of sum of 15 PAHs (µg g-1 dry weight) varied from 1.7 (bedroom in detached house in rural area) to 67.9 µg g-1 (room with heavy smokers in detached house in the city), and were dominated by 3-ring (6.89-57.1%) and 2-ring compounds (5.05-48.3%). The result of cluster analysis (CA) suggested two distinct groups of PAHs. The dominant PAH source was found to be mixed petrogenic and pyrogenic consisting of a mixture of cooking, smoking, heating and neighbouring traffic activities.

Biomolecular Evidence of Silk from 8,500 Years Ago.

Pottery, bone implements, and stone tools are routinely found at Neolithic sites. However, the integrity of textiles or silk is susceptible to degradation, and it is therefore very difficult for such materials to be preserved for 8,000 years. Although previous studies have provided important evidence of the emergence of weaving skills and tools, such as figuline spinning wheels and osseous lamellas with traces of filament winding, there is a lack of direct evidence proving the existence of silk. In this paper, we explored evidence of prehistoric silk fibroin through the analysis of soil samples collected from three tombs at the Neolithic site of Jiahu. Mass spectrometry was employed and integrated with proteomics to characterize the key peptides of silk fibroin. The direct biomolecular evidence reported here showed the existence of prehistoric silk fibroin, which was found in 8,500-year-old tombs. Rough weaving tools and bone needles were also excavated, indicating the possibility that the Jiahu residents may possess the basic weaving and sewing skills in making textile. This finding may advance the study of the history of silk, and the civilization of the Neolithic Age.

Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori.

The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography-tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study.

Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy.

The identification of ancient silk is of great importance in both archaeology and academia. In the present work, a specific antibody having the characteristics of low cost, easy operation and extensive applicability was developed directly through immunizing rabbits with complete antigen (silk fibroin, SF). Then, antibody-based immunoassays, i.e. enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM), were established and conducted in tandem to identify the corresponding protein in ancient silks. The anti-SF antibody exhibits high sensitivity and specificity for the identification of modern and ancient silks. The detection limit of the ELISA method is about 0.1 ng/mL, and no cross-reactions with other possible interference antigens have been noted. IFM makes it possible to localize target proteins in archaeological samples, and also ensure the reliability of the ELISA results. Based on these advantages, immunological techniques have the potential to become powerful analytical tools at archaeological sites and conservation science laboratories.

Effect of mechanical deformation on the structure of regenerated Bombyx mori silk fibroin films as revealed using Raman and infrared spectroscopy.

To better understand the effect of mechanical stress during the spinning of silk, the protein orientation and conformation of Bombyx mori regenerated silk fibroin (RSF) films have been studied as a function of deformation in a static mode or in real time by tensile-Raman experiments and polarization modulation infrared linear dichroism (PM-IRLD), respectively. The data show that either for step-by-step or continuous stretching, elongation induces the progressive formation of ß-sheets that align along the drawing axis, in particular above a draw ratio of ~2. The formation of ß-sheets begins before their alignment during a continuous drawing. Unordered chains were, however, never found to be oriented, which explains the very low level of orientation of the amorphous phase of the natural fiber. Stress-perturbed unordered chains readily convert into ß-sheets, the strain-induced transformation following a two-state process. The final level of orientation and ß-sheet content are lower than those found in the native fiber, indicating that various parameters have to be optimized in order to implement a spinning process as efficient as the natural one. Finally, during the stress relaxation period in a step-by-step drawing, there is essentially no change of the content and orientation of the ß-sheets, suggesting that only unordered structures tend to reorganize.

Dragline silk: a fiber assembled with low-molecular-weight cysteine-rich proteins.

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

Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors.

Miniature bioreactors under parallel fed-batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale-up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed-batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred-tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed-batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD600 ) of 114-133 and final DCW concentrations of 69-70 g L(-1) . The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.

Post-secretion processing influences spider silk performance.

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 ß-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.

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

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.

The absence of detectable fetal microchimerism in nontransgenic goats (Capra aegagrus hircus) bearing transgenic offspring.

Regulations for the disposal of genetically engineered animals are strict due to concern for their inappropriate introduction into the food chain, and of the possible public health and environmental impacts of these organisms. Nontransgenic animals that give birth to transgenic offspring are treated as if they are transgenic due to concern of fetal cells crossing the placental barrier and residing in the mother (fetal-maternal microchimerism). Determining whether or not fetal-fetal or fetal-maternal transfer of DNA or cells occurs during caprine gestation is critical to effectively protect the public without culling animals that pose no risk. Additionally, fetal-maternal transfer, should it exist in the goat, could contraindicate the rebreeding of nontransgenic dams due to the possible transfer of fetal cells from 1 pregnancy to the fetus of subsequent pregnancies. Fetal-maternal transfer in Capra hircus has not been reported in the literature, although it has been reported in another ruminant, Bos taurus. We examined blood from nontransgenic dams that carried transgenic offspring using a PCR method sensitive enough to detect the presence of a spider silk transgene to a 1:100,000 dilution. At this sensitivity, we did not detect the occurrence of fetal-maternal transfer in 5 nontransgenic dams. Likewise, fetal-fetal transfer was not observed from a transgenic to a nontransgenic twin in utero. To test tissue-specific expression of the silk transgene, proteins purified from standard necropsy tissue from a lactating transgenic dam were examined by Western blot analysis. Silk protein expression was only observed in mammary tissue consistent with the tissue specificity of the ß-casein promoter used in the transgenic construct. We report evidence collected from a limited caprine breeding pool against transfer of transgenes in utero from fetus to dam and fetus to fetus. In addition, we show evidence that the ß-casein promoter in our expression construct is not expressed ectopically as previously suggested. These results suggest that transgene transfer in utero does not occur, but further study is warranted with a larger sample group to confirm these results.

Synchrotron FTIR microspectroscopy of single natural silk fibers.

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 ß-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 ß-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.

An investigation into silk fibroin conformation in composite materials intended for drug delivery.

Regenerated silk fibroin (SF) is a promising biomaterial to design drug delivery systems. To guarantee satisfactory prolonged release of loaded drugs, the native ß-sheet conformation of SF is generally induced by a final curing which can determine instability of the loaded drug. This work aimed to investigate the influence on SF conformation of the addition of hydrophilic polymers, namely poloxamer 188 (PEO), a range of poly(ethylenglycol) (PEG)and poly(vinyl pyrrolidone) (PVP) and drying conditions, namely spray-drying or evaporation at 60 °C. DSC data on spray-dried products indicated that SF in composite materials was in the random coil conformation. ATR-FTIR spectroscopy with Fourier self-deconvolution of the amide I band revealed that SF in spray dried products was partially organized in the ß-sheet structure only in presence of PEG4000. Both DSC and ATR-FTIR spectra registered on composite materials obtained by the slowest evaporation method indicated that all hydrophilic polymers favoured the ß-sheet conformation. This feature was attributed to the formation of H-bonds with the tyrosine residues of the semicrystalline region in SF. In conclusion, this approach to prepare of SF/hydrophilic polymer composites at slow evaporation rate leads to water insoluble materials which could be used in the development of drug delivery systems.

Characterization of tyrosine-rich Antheraea pernyi silk fibroin hydrolysate.

Antheraea pernyi silk fibroin (SF) hydrolysate were characterized using UV-VIS spectrometer, amino acid composition and heavy metal contents to explore its potential sources for food or cosmetic additives. The hydrolyzed A. pernyi SF was separated into two parts: (a) SFA, alanine-rich fraction and (b) SFB, tyrosine-rich fraction. SFB exhibited strong absorption peaks at 210 and 280 nm due to the presence of the tyrosine. Heavy metal analysis showed that arsenic and mercury did not detect. Other heavy metals, which includes lead, cadmium, etc., were recorded only a trace amount. Therefore, A. pernyi SF hydrolysate could be safely used as sources of food, cosmetic and pharmaceuticals.

The effect of genetically engineered spider silk-dentin matrix protein 1 chimeric protein on hydroxyapatite nucleation.

Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of Nephila clavipes fused to the carboxyl terminal domain of dentin matrix protein 1 (CDMP1). The objective was to exploit the self-assembly and material properties of silk proteins with controlled hydroxyapatite (HA) formation from CDMP1, for novel biomaterial composites. The purified recombinant protein retained native-silk like self-assembly properties and beta-sheet structure when formed into films and treated with methanol. When the chimeric protein in solution was incubated with CaCl(2,) the secondary structure shifted from random coil to alpha-helix and beta-sheet, due to the interactions between the CDMP1 domain and Ca(2+). The control protein without the CDMP1 domain did not undergo a similar transition. Films formed from the recombinant protein were mineralized using simulated body fluids and induced the formation of calcium-deficient carbonated HA, Ca(10)(PO(4))(6)(OH)(2) based on SEM, EDS, FTIR and TEM analysis. This mineral phase was not formed on the films formed from the control spider silk protein without the CDMP1 domain. Considering the osteoconductivity of HA and the novel material features of spider silks, these new hybrid systems offer potential as biomaterials for a number of potential applications.

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution.

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