Search

Silk proteins, as natural macromolecules, have extensive applications in biomaterials and biomedicine. In the silkworm, the expression of silk protein genes is negatively associated with ecdysone during the molt stage, while it is positively correlated with juvenile hormone during the intermolt stage. In our previous study, overexpression of an isoform Z2 of Broad Complex (BmBrC-Z2), an ecdysone early response factor, significantly reduced the expression of silk protein genes. However, the underlying regulatory mechanism remains unclear. In this study, we conducted transcriptomic analysis and found that overexpressing BmBrC-Z2 significantly upregulated the expression level of multiprotein bridging factor 2 (BmMBF2), an inhibitor of fibroin heavy chain (FibH). Further investigations revealed that BmBrC-Z2 directly regulated BmMBF2 by binding to cis-regulatory elements, as demonstrated using Dual-Luciferase Reporter Gene Assay, EMSA, and ChIP-PCR assay. Additionally, when using the CRISPR/Cas9 system to knock out BmMBF2, silk protein genes were significantly upregulated during the molt stage of mutant larvae. These findings uncover the negative regulation of silk protein synthesis by the ecdysone signaling cascade, specifically through the manipulation of BmMBF2 expression during the molt stage. This study enhances to our understanding of the temporal regulatory mechanism governing silk protein synthesis and offers a potential strategy for improving silk yield., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest associated with this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The silkworm is an incredibly valuable insect that produces silk through its silk gland. Within this organ, Fibroinase has been identified and named due to its ability to fibroin degradation. The expression of Fibroinase in the silk gland significantly increases during the larval-pupal stage, which might be associated with the degeneration of the silk gland. In this study, Fibroinase was overexpressed and knocked down specifically both in the middle and posterior silk glands, respectively, using transgenic technology. The investigation of silk gland development in these transgenic silkworms showed that Fibroinase plays a direct role in accelerating silk gland degeneration. The staining analyses performed in the silk glands of transgenic silkworms suggest that Fibroinase is involved in the processes of autophagy and apoptosis during silk gland degeneration. Further experiments demonstrated that Fibroinase, acting as a lysosomal regulator, negatively regulates autophagy via the mTOR (mechanistic target of rapamycin) pathway. Moreover, during apoptosis, Fibroinase could activate Caspase3 by increasing the activity of BmCaspase1, ultimately accelerating the apoptosis process. These findings enhance our understanding of the physiological role of Fibroinase in promoting silk gland degeneration, which plays a role in breaking down proteins in the silk gland and coordinating the regulation of autophagy and apoptosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2, differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Silkworm fibroins are natural proteinaceous macromolecules and provide core mechanical properties to silk fibers. The synthesis process of fibroins is posterior silk gland (PSG)-exclusive and appears active at the feeding stage and inactive at the molting stage. However, the molecular mechanisms controlling it remain elusive. Here, the silk gland's physiological and nuclear proteomic features were used to characterize changes in its structure and development from molting to feeding stages. The temporal expression profile and immunofluorescence analyses revealed a synchronous transcriptional on-off mode of fibroin genes. Next, the comparative nuclear proteome of the PSG during the last molting-feeding transition identified 798 differentially abundant proteins (DAPs), including 42 transcription factors and 15 epigenetic factors. Protein-protein interaction network analysis showed a "CTCF-FOX-HOX-SOX" association with activated expressions at the molting stage, suggesting a relatively complex and multifactorial regulation of the PSG at the molting stage. In addition, FAIRE-seq verification indicated "closed" and "open" conformations of fibroin gene promoters at the molting and feeding stages, respectively. Such proteome combined with chromatin accessibility analysis revealed the detailed signature of protein factors involved in the temporal regulation of fibroin synthesis and provided insights into silk gland development as well as silk production in silkworms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the α-CA class in the silk gland, which is a Zn 2+ dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO 2 to HCO 3 - , thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved. Tissue expression profiling showed that BmeryCA had relatively high expression levels in hemolymph and epidermis but is barely expressed in the posterior silk gland (PSG). By specifically overexpressing BmeryCA in the PSG, we generated transgenic silkworms. Ion-selective microelectrode (ISM) measurements demonstrated that specifically overexpression of BmeryCA in the PSG led to a shift in pH from weakly alkaline to slightly neutral conditions. Moreover, the resultant PSG-specific BmeryCA overexpression mutant strain displayed a significant increase in both silk yield and silk fiber mechanical properties. Our research provided new insights into enhancing silk yield and improving the mechanical properties of silk fibers., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

The silkworm is a lepidopteran domesticated from the wild silkworm, mostly valued for its efficient synthesis of silk protein. This species' ability to spin silk has supported the 5500-year-old silk industry and the globally known "Silk Road", making the transformation of mulberry leaves into silk of great concern. Therefore, research on the silk-related genes of silkworms and their regulatory mechanisms has attracted increasing attention. Previous studies have revealed that domestic silk gland cells are endoreduplication cells, and their high-copy genome and special chromatin conformation provide conditions for the high expression of silk proteins. In this study, we systematically investigate the expression pattern of eukaryotic initiation factors (eIFs) and identified the eIF6 as a eukaryotic translation initiation factor involved in the synthesis of silk proteins. We generated an eIF6 gene deletion mutant strain of silkworm using the CRISPR/Cas9 system and investigated the function of eIF6 in silk gland development and silk protein synthesis. The results showed that deletion of eIF6 inhibited the individual development of silkworm larvae, inhibited the development of silk glands, and significantly reduced the cocoon layer ratio. Therefore, we elucidated the function of eIF6 in the development of silk glands and the synthesis of silk proteins, which is important for further elucidation of the developmental process of silk glands and the mechanism underlying the ultra-high expression of silk proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

HSA is considered a versatile natural cargo carrier with multiple bio-functions and applications. However, insufficient supply of HSA has limited widespread use. Although various recombinant expression systems had been applied to produce the rHSA to overcome the limited resource, cost-effective and large scale production of rHSA remains a challenge. Herein, we provide a strategy for the large-scale and cost-effective production of rHSA in cocoons of transgenic silkworms, achieving a final 13.54 ± 1.34 g/kg of rHSA yield in cocoons. rHSA was efficiently synthesized and stable over the long-term in the cocoons at room temperature. Artificial control of silk crystal structure during silk spinning significantly facilitated rHSA extraction and purification, with 99.69 ± 0.33 % purity and a productivity of 8.06 ± 0.17 g rHSA from 1 kg cocoons. The rHSA had the same secondary structure to natural HSA, along with effective drug binding capacity, biocompatibility, and bio-safe. The rHSA was successfully evaluated as a potential substitute in serum-free cell culture. These findings suggest the silkworm bioreactor is promising for large-scale and cost-effective production of high quality rHSA to meet the increased worldwide demand., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Body size and silk protein synthesis ability are two crucial aspects of artificial selection in silkworm breeding; however, the role of genes in both pathways remains unknown. To determine whether let-7 microRNA could regulate larval development and silk gland growth simultaneously, we designed a guide RNA to edit let-7 using the CRISPR/Cas9 system. The indels predominantly appeared in the let-7 seed region, and the vast majority of the mutations were small-fragment deletions. Loss of let-7 function prolonged the fifth larval period, and substantially increased body weight during the wandering stage, but it resulted in developmental arrest during the pupal-moth transition. let-7 systemic knock down promoted silk gland growth and increased silk yield by >50 %, with efficiency significantly higher than in tissue-specific edited individuals. Hormone signaling and cell cycle pathway genes were activated in different patterns in the body and silk gland, implying that let-7 may regulate different target genes to play role in tissue growth. In summary, we first report that conditional knock down let-7 promoting the simultaneous growth of body and silk gland, greatly improve silk yield in the silkworm., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Silk gland genes of Bombyx mori can have strict spatial expression patterns, which impact their functions and silk quality. However, our understanding of their regulation mechanisms is currently insufficient. To address this, the middle silk gland (MSG) and posterior silk gland (PSG) of the silkworm were investigated. Gene ontology annotation showed that spatially specific expressed genes were involved in the formation of H3k9me and chromatin topology. Chromatin conformation data generated by Hi-C showed that the topologically associated domain boundaries around FibL and Sericin1 genes were significantly different between MSG and PSG. Changes in chromatin conformation led to changes in chromatin activity, which significantly affected the expression of nearby genes in silkworm. Chromatin accessibility regions of MSG and PSG were analyzed using FAIRE-seq, and 1006 transcription factor motifs were identified in open chromatin regions. Furthermore, the spatial-specific expression patterns of silk gland genes were mainly associated with homeobox-contained transcription factors, such as POU-M2, which was specifically bound and relatively highly expressed in the MSG. The regulatory network mediated by POU-M2 regulated most of the spatial-specific expressed genes in MSG, such as ADH1. These results can aid in improving silk performance, optimizing silkworm breeding, and improving the gene spatial regulatory model research for insects., Competing Interests: Declaration of competing interest All authors disclosed no relevant relationships., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Silkworms spin different silks at different growth stages for specific purposes. The silk spun before the end of each instar is stronger than that at the beginning of each instar and cocoon silk. However, the compositional changes in silk proteins during this process are unknown. Consequently, we performed histomorphological and proteomic analyses of the silk gland to characterize changes from the instar end to the next instar beginning. The silk glands were collected on day 3 of third- and fourth-instar larvae (III-3 and IV-3) and the beginning of fourth-instar larvae (IV-0). Proteomic analysis identified 2961 proteins from all silk glands. Silk proteins P25 and Ser5 were significantly more abundant in III-3 and IV-3 than in IV-0, and many cuticular proteins and protease inhibitors increased significantly in IV-0 compared with III-3 and IV-3. This shift may cause mechanical property differences between the instar end and beginning silk. Using section staining, qPCR, and western blotting, we found for the first time that silk proteins were degraded first and then resynthesized during the molting stage. Furthermore, we revealed that fibroinase mediated the changes of silk proteins during molting. Our results provide insights into the molecular mechanisms of silk proteins dynamic regulation during molting., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tissue/organ remodeling and cells fate determination play key roles in the life cycle of animals. However, they are still poorly understood in insects, especially in the silkworm. The anterior silk gland (ASG) of the silkworm is essential for the formation and performance of silk fibers, but the regulatory mechanism of ASG remodeling and cells fate determination is less known. Here we found that silencing of POUM2 caused shorter ASG length, intimal structural defects, silkworm spinning failure, and the resultant naked pupae death, but cells number was not affected. Cells staining showed that DNA endoreduplication was not affected in the ASG. Transmission electron microscopy and chitin staining showed cuticle proteins and chitin were greatly reduced in the ASG during the molting period. Transcriptional analysis showed the expression profiles of cuticle proteins and chitin synthase were similar to that of POUM2 during the molting period, and POUM2 down-regulation reduced the expression of cuticle proteins, chitin synthase, autophagy and apoptosis-related genes. While the phenotype resulting from POUM2 over-expression was similar to that of POUM2 down-regulation. Cells staining revealed marked cells apoptosis with cells number reduction and inhibition of DNA endoreduplication in the ASG. Transcriptional analysis showed the expression of autophagy and apoptosis-related genes, and some cuticle proteins and chitin synthase were significantly up-regulated. The results suggest that POUM2 homeostasis regulates ASG intimal remodeling and cells fate, thus affecting ASG development, silkworm spinning and metamorphosis. Our studies not only offer potential molecular targets for genetic improvement of silk performance and molecular breeding of the silkworm, but also provide new insights into POU factor-mediated tissue remodeling and cells fate determination in insects., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Silkworm spins silk fibers to make the cocoon to protect the pupa from predators and pathogenic microbes. To understand the defense mechanism of the cocoon, many antimicrobial proteins are currently identified. The functionality of these proteins is studied, including protease inhibitors and seroins. Protease inhibitors are incredibly variable in sequences and domains, and most of them contain multiple pairs of disulfide bonds. Thereby they have stable structures and activities. Seroins have two motifs: the proline-rich N-terminal motif and the sequence conserved C-terminal motif. Protease inhibitors mainly play antifungal roles, whereas seroins have broad-spectrum antimicrobial activities against bacteria, fungi and viruses. These antimicrobial proteins show higher abundance in the sericin layers than in the fibroin layer and are more abundant in the outer cocoon layer than in the inner cocoon layer. Besides silk proteins, the silkworm cocoon also contains small amounts of non-protein antimicrobial components such as organic acids, alkaloids, flavonoids, and heterocyclic compounds. This review describes the extraction methods, expression pattern, microbiostatic mechanism, application fields and advantages of the antimicrobial components in the silkworm cocoon. The in-depth understanding of antimicrobial silk components will help us improve the processing technology of cocoons and expand the application fields of the cocoons., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rearing silkworms (Bombyx mori) using formula feed has revolutionized traditional mulberry feed strategies. However, low silk production efficiencies persist and have caused bottlenecks, hindering the industrial application of formula feed sericulture. Here, we investigated the effects of formula feed amino acid composition on silk yields. We showed that imbalanced amino acids reduced DNA proliferation, decreased Fib-H, Fib-L, and P25 gene expression, and caused mild autophagy in the posterior silk gland, reducing cocoon shell weight and ratio. When compared with mulberry leaves, Gly, Ala, Ser, and Tyr percentages of total amino acids in formula feed were decreased by 5.26%, while Glu and Arg percentages increased by 9.56%. These changes increased uric acid and several amino acids levels in the hemolymph of silkworms on formula feed. Further analyses showed that Gly and Thr (important synthetic Gly sources) increased silk yields, with Gly increasing amino acid conversion efficiencies to silk protein, and reducing urea levels in hemolymph. Also, Gly promoted endomitotic DNA synthesis in silk gland cells via phosphoinositide 3-kinase (PI3K)/Akt/target of rapamycin (TOR) signaling. In this study, we highlighted the important role of Gly in regulating silk yields in silkworms., (Copyright © 2022. Published by Elsevier B.V.)

UV radiation causes excess production of melanin as a result of hyperpigmentation and skin disorders. Silk sericin exhibited bioactivities to skin and inhibited UV-induced phototoxicity and melanogenesis in skin cells; however, the mechanism related to sericin against UV-induced melanogenesis has not been investigated. This study aimed to investigate the protective effects of Thai silk sericins against UVA-induced phototoxicity and melanogenesis and their related mechanisms. Thai silk sericins exhibited cytoprotective effects against UV-induced toxicity in human primary melanocytes by attenuation of cytotoxicity, intracellular ROS generation, and mitochondrial potential impairment. Pre- and post-treatment with sericin significantly inhibited melanin synthesis and tyrosinase activity against UVA exposure. In addition, sericin S2 could reduce the basal melanin content in zebrafish embryos. The proteomic analysis demonstrated that Thai silk sericins altered the protein expression in melanocytes especially proteins related to stress, inflammatory, cytokine stimulation, cell proliferation, and cell survival processes that contribute to cytoprotective effect and inhibitory effect on melanogenesis of sericin. Moreover, we demonstrated the novel mechanism of Thai silk sericins in inhibiting UVA-induced melanogenesis via increasing BMP4 expression in MAPK/ERK signaling pathway. These evidences support the potential use of Thai silk sericins in prevention of hyperpigmentation in skin disorders especially after UVA exposure., (Copyright © 2021. Published by Elsevier B.V.)

The unique physiochemical properties and the porous network architecture of hydrogel seek the attention to be explored in broad range of fields. In the last decade, numerous studies on the development of enzymatically cross-linked hydrogels have been elucidated. Implementing enzyme based cross-linking for fabrication of biomaterials over other cross-linking methods harbor various advantages, especially hydrogels designed using laccase exhibits mild reaction environment, high cross-linking efficiency and less toxicity. To our knowledge this is the first report reviewing the formulation of laccase mediated cross-linking for hydrogel preparation. Here, laccase catalyzed synthesis of hydrogel using polysaccharide viz. arabinoxylan, sugar beet pectin, galactomannan, chitosan etc. and proteins namely soy protein, gelatin, silk fibroin were discussed on highlighting their mechanical properties and its possible field of application. We have summarized the role of phenolic acids in laccase mediated cross-linking particularly ferulic acid which is a component of lignocellulose, serving cell rigidity via cross-linkage. The review also discusses on various biomedical applications such as controlled protein release, tissue engineering, and wound healing. It is anticipated that this review will give a detailed information on different laccase mediated reaction strategies that can be applied for the synthesis of various new biomaterials with tailor made properties., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fibroin of the silkworm consists of fibroin heavy chain (Fib-H) with hydrophobic intermediate repeats flanked by hydrophilic N and C terminal domains (NTD and CTD, respectively), fibroin light chain (Fib-L), and P25. However, the respective roles of each polypeptide in silk processing remain largely unknown. Here, a series of transgenic silkworms with different fusion gene expression cassettes were created in order to selectively express different fluorescent fusion proteins in silk glands. The roles of different components in silk processing were investigated via observing and analyzing the movement and distribution of these proteins in the silk gland and in cocoon silk. The data showed that hydrophilic NTDs were distributed on the surface of micelles, providing sufficient electrostatic repulsion to prevent premature crystallization of silk proteins. Hydrophilic CTD==Ls ("==" represents the disulfide bond) were located on the inner layer of micelles to control the solubility of large micelles. The results presented here elucidated the underlying mechanisms of silkworm silk processing in vivo. This is significant for the development of artificial spinning technology, novel silk biomaterials, and silk gland expression systems., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A silkworm cocoon contains several antimicrobial proteins such as protease inhibitors and seroins to provide protection for the enclosed pupa. In this study, we identified a new Bombyx mori phosphatidylethanolamine-binding protein (BmPEBP) with antimicrobial activity in the cocoon silk using semi-quantitative and quantitative RT-PCR, western blotting, and immunofluorescence. The results indicated that BmPEBP was synthesized in the middle silk gland and secreted into the sericin layer of the cocoon silk. Functional analysis showed that BmPEBP could inhibit the spore growth of four types of fungi, Candida albicans, Saccharomyces cerevisiae, Beauveriabassiana, and Aspergillus fumigates, by binding to the fungal cell membrane. Investigation of the interaction of BmPEBP with membrane phospholipids revealed that the protein showed a strong binding affinity to phosphatidylethanolamine, weak affinity to phosphatidylinositol, and no affinity to phosphatidylserine or phosphatidylcholine. Circular dichroism spectroscopy showed that binding to phosphatidylethanolamine caused conformational changes in the BmPEBP molecule by reducing β-sheet formation and inducing the appearance of an α-helix motif. We speculate that BmPEBP performs antifungal function in the cocoon silk through interaction with phosphatidylethanolamine in the fungal membrane., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A powerful system for studying protein aggregation, particularly rapid self-assembly, is spider silk. Spider silks are proteinaceous and silk proteins are synthesized and stored within silk glands as liquid dope. As needed, liquid dope is near-instantaneously transformed into solid fibers or viscous adhesives. The dominant constituents of silks are spidroins (spider fibroins) and their terminal domains are vital for the tight control of silk self-assembly. To better understand spidroin termini, we used target capture and deep sequencing to identify spidroin gene sequences from six species representing the araneoid families of Araneidae, Nephilidae, and Theridiidae. We obtained 145 terminal regions, of which 103 are newly annotated here, as well as novel variants within nine diverse spidroin types. Our comparative analyses demonstrated the conservation of acidic, basic, and cysteine amino acid residues across spidroin types that had been proposed to be important for monomer stability, dimer formation, and self-assembly from a limited sampling of spidroins. Computational, protein homology modeling revealed areas of spidroin terminal regions that are highly conserved in three-dimensions despite sequence divergence across spidroin types. Analyses of our dense sampling of terminal regions suggest that most spidroins share stabilization mechanisms, dimer formation, and tertiary structure, despite producing functionally distinct materials., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In tissue engineering, chemical crosslinking is widely used for conjugating two or more biomaterials to mainly control biodegradability and strength. For example, Thai silk fibroin/gelatin scaffold will offer mechanical strength from Thai silk fibroin and cell attraction from gelatin. However, chemical crosslinking requires crosslinking agent which could potentially pose negative impact from remaining trace amount of chemicals especially in medical application. Here we present an alternative approach to chemical crosslinking-a balance electrostatic blending approach. In this approach, two opposite charge biomaterials were selected for blending, with different ratios. Both materials were bound together with electrostatic force. The maximum binding was achieved when mixture electric potential approaches zero. In this work, we compared this approach with traditionally chemical crosslinking in terms of physical appearance, binding effectiveness, mechanical strength (in dry/wet conditions), in vitro biodegradation, and cell proliferation. We found that 50/50 weight ratio of Thai silk fibroin/gelatin scaffold had almost comparable properties to chemical crosslinked scaffold. It has similar appearance, binding effectiveness, and affinity for cell proliferation. For mechanical properties, even this approach yields lower dry compressive modulus compared with chemical crosslinking. But in wet condition, the compressive modulus from both methods is similar. However, the biodegradation time of non-crosslinked scaffolds is slightly faster than that of chemical crosslinked ones. These results demonstrate that a balance electrostatic approach is an alternative approach to chemical crosslinking when there is a concern of remaining trace amount of crosslinking agent in medical application., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Silk fibers produced by undomesticated wild insects belonging to the Saturniidae family have unique properties compared to the commonly used silks. Insects belonging to the Saturniidae family are one of the largest moths found throughout the world, produce large cocoons and are easier to rear than B. mori. In this research, we have characterized the morphology and tensile properties of silks produced by eight wild insects that belong to the Saturniidae family. Fibers produced by Saturniidae insects such as C. hercuels have properties similar to that of B. mori silk whereas fibers produced by Copaxa multifenestrata have inferior properties than B. mori or the common wild silks. In addition, the tensile properties of the fibers vary considerably between insects. Fibers with fineness ranging from 1.5 to 7.8denier and breaking tenacity ranging from 0.9 to 5g per denier are produced by the Saturniidae insects. Identifying the unique properties of Saturniidae silks such as tensile properties, yield of silk from the cocoons and ability to grow under different environments will help to evaluate the potential of rearing the wild insects for commercial production of silk for textile, medical and other applications., (2010 Elsevier B.V. All rights reserved.)

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.

The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.

Currently, studies on major ampullate spidroin 1 (MaSp1) genes of non-orb weaving spiders are few, and it is not clear whether genes of these organisms exhibit the same characteristics as those of orb-weavers. In addition, many studies have proposed that MaSp1 might be a single gene with allelic variants, but supporting evidence is still lacking. In this study, we compared partial DNA and amino acid sequences of MaSp1 cloned from different spider guilds. We also cloned partial MaSp1 sequences from genomic DNA and cDNA of the same individuals of spiders using the same primer combination to see if different molecular forms existed. In the repetitive region of partial MaSp1 sequences obtained, GGX, GA and poly-A motifs were present in all Araneomorphae and Mygalomorpae species examined. An extreme similarity in MaSp1 non-repetitive portions was found in sequences of ecribellate, cribellate and Mygalomorphae web-builders and such a result suggested that this sequence might exhibit an important function. A comparison of sequences amplified from the same individual showed that substitutions in amino acids occurred in both repetitive and non-repetitive regions, with a much higher variation in the former. These results suggest that the MaSp1 of Araneomorphae spiders exhibits several forms in an individual spider and it might be either a multiple gene or a single gene with a multiple exon/intron organization.