Structural insight on the liquid silk from the middle silk gland of non-mulberry silkworm Antheraea assamensis.

This study highlights the preliminary characterization of liquid silk from the middle silk gland (MSG) along with the in-silico analysis of the sericin protein of a less explored non mulberry silkworm Antheraea assamensis which is endemic to the North Eastern region of India. Various biophysical methods have been applied to elucidate the conformational patterns of the liquid silk present inside the MSG without removing the sericin layer. This will help us to know the actual features of the in vivo transitional status of the silk in the MSG which travel towards the anterior silk gland (ASG) prior to spinning. The SDS PAGE analysis represented the existence of the both fibroin and sericin bands in the sample. The structural pattern of the MSG liquid silk as revealed by various methods denoted the occurrence of ß-sheet component along with some random coil and ß-turn components which in turn suggests the transitional state of the liquid silk attributed to the existence of both the crystalline and amorphous contents. The thermo gravimetric study and the aggregation behavior analysis results proposed the occurrence of intermolecular hydrogen bonding between the sericin and fibroin in the MSG. This study will sensitize the better understanding of the behavior of the liquid silk in the MSG of non-mulberry silkworm A. assamensis and will open avenues for various application-based studies of this silk.Communicated by Ramaswamy H. Sarma.

Composition and in silico structural analysis of fibroin from liquid silk of non-mulberry silkworm Antheraea assamensis.

Silk is spun from the liquid precursor known as liquid silk secreted from the posterior part and stored in the silk gland lumen with occurrence of many momentary events. The liquid silk in the silk gland is transformed to the spun silk fibre. In this study the elucidation of the protein components of liquid silk from the posterior part of the silk gland (PSG) of saturniid silkworm Antheraea assamensis along with its structural characterization has been reported. The 3D model of the N-terminal amorphous portion with some repeat crystalline motifs (19-255) of core protein fibroin has also been constructed. 1D and 2D electrophoresis revealed the homo-dimeric structure of the silk protein. Secondary structure analysis by Circular dichroism, FTIR spectroscopy showed α helical structural component as predominant conformation in the liquid silk. The crystalline structure investigated through X ray diffraction (XRD) analysis also revealed the presence of less ordered amorphous α helical conformation in the liquid silk. The 3D structural model proposed of the residues from 19 to 255 has revealed structural stability throughout the molecular dynamics simulation process. This study will provide the detailed structural information and in silico analysis of the core protein present in the liquid silk of PSG.

Novel non-mulberry silk fibroin nanoparticles with enhanced activity as potential candidate in nanocarrier mediated delivery system.

Silk fibroin (SF) is well known for its excellent biocompatible properties facilitating its application in the field of biomedical engineering through different biomaterial fabrications in the recent era. Here in this study, novel nanoparticles from non-mulberry SF of Antheraea assamensis were fabricated, characterized and evaluated for its applicability as nanocarrier. Fabricated nanoparticles were initially compared with prevailing SF nanoparticles from Bombyx mori. Fabricated A. assamensis silk fibroin nanoparticles (AA-SFNps) were found to be lesser in size (80-300 nm in diameter) than B. mori silk fibroin nanoparticles (BM-SFNps) (120-500 nm in diameter). When checked for stability, AA-SFNps were found to be more stable than BM-SFNps in biological media. FTIR and XRD studies revealed persistence of structural properties even after fabrication. TGA and DSC studies showed AA-SFNps to be thermally more stable than BM-SFNps without any cytotoxicity (MTT assay). On loading with model drug Doxorubicin hydrochloride (DOX), AA-SFNps exhibited an encapsulation efficiency of 94.47% with 11.81% loading of the anticancer drug. Cumulative release study revealed highest percentage release of DOX (42.1 ± 0.4%) at pH 5.2 on day 7 in comparison to pH 7.4 and 8.0. Sustained release profile of the DOX loaded AA-SFNps (AA-SFNps-DOX) was clearly reflected and it was found to be highly cytotoxic against triple negative MDA-MB-231 cells in comparison to free DOX at different time points. Overall, this study showed the efficacy of the AA-SFNps as a nanocarrier for future drug delivery applications.

Variations in the Metallic Ion Concentration in the Silk Gland and Cocoon of Silkworm Antheraea assamensis helfer.

The natural spinning process in silkworms involves the conformation transition of the liquid silk protein present in the silk gland to make fiber. This conformation transition is influenced by different factors, and some studies suggested that changes in the metallic ion concentrations is one of them (Zhou et al. 2005). This study investigated the changes in the metallic ion compositions in the silk glands (before and during spinning) and cocoons of non-mulberry silkworm Antheraea assamensis helfer. Intact silk glands were dissected from mature 5th instar A. assamensis larvae. The glands were rinsed with deionized water and divided into five divisions: posterior silk gland (PSG), middle silk gland (MSG), anterior silk gland (ASG), posterior middle (PM) and anterior middle (AM) division of silk gland. Cocoon pieces and the gland parts were dried and digested in acid mixture to quantify the metallic contents in an atomic absorption spectrophotometer (Shimadzu, AA7000). We determined seven metals (Na, K, Mg, Ca, Cu, Zn, Fe) present in the different parts of the secretory pathway as well as in the fibers of A.assamensis. Our results suggested that the concentrations of Mg, Ca, Na, and K were more abundant in the gland than the Cu and Zn. Fe concentration was found comparatively less in amount in the gland. Amount of Ca found to be higher in the cocoons. The differences in the metallic ion concentrations in the gland parts before and during spinning suggested the possibility of their role in the formation of silk thread from luminal silk.

Genetic diversity and phylogeny analysis of Antheraea assamensis Helfer (Lepidoptera: Saturniidae) based on mitochondrial DNA sequences.

Antheraea assamensis Helfer, popularly known as Muga silkworm, the golden silk producer of northeast India is economically important and unique among the Saturniid silkworms. In this study, the genetic diversity and phylogeny of semidomesticated and wild morphs of Muga silkwormcollected from different geographical locations of northeast India were investigated based on the sequences of five mitochondrial loci, i.e. 12S rRNA, 16S rRNA, CoxI, Cytb and CR. All the five mitochondrial loci showed a strong bias towards higher 'A' and 'T' contents. Transitional substitutions were found to be more than the transversional substitutions. The rate of nucleotide substitution and average genetic divergence were found to be highest in CR sequences and lowest in 12S rRNA gene sequences among the morphs of Muga silkworm. The morphs collected from same geographical area had identical 12S rRNA, 16S rRNA, CoxI and Cytb gene sequences. Moreover, the 12S rRNA and 16S rRNA gene sequences of somesemi-domesticated and wild morphs collected from different geographical locations were also found to be similar. In the phylogenetic trees generated based on themitochondrial loci, mixing of semi-domesticated and wild morphs was observed as they shared the same group. The information generated in this study will help in formulating strategies to conserve the natural biodiversity present among these unique silkworms in northeast India. In addition, this will be useful in identifying diverse morphs of Muga silkworm, which will help in effective breeding programmes to improve its productivity.

Analysis of genetic diversity and phylogeny of Philosamia ricini (Lepidoptera: Saturniidae) by using RAPD and internal transcribed spacer DNA1.

The Indian Eri silkworm, Philosamia ricini Hutt, a commercial silk producing insect, is believed to have originated in the Brahmaputra valley of Assam. In this study, the genetic diversity and phylogenetic relationships of six morphs of Eri silkworm viz. white plain, white zebra, white spotted, blue plain, blue zebra and blue spotted collected from different geographical locations of North-East India were investigated by using random amplified polymorphic DNA (RAPD) and the first internal transcribed spacer region (ITS1). This study revealed a low genetic diversity among the morphs of Eri silkworm. Twenty-eight random primers generated 199 bands. Out of these, 112 were polymorphic (56.28%) with an average of 7.1 bands per primer. The genetic similarity matrix ranged from 0.56 to 0.99. The morphs collected from same geographical area shared the same cluster in the dendrogram. The genetic diversity in case of ITS1 sequences (2.19%) was found to be less as compared to RAPD. The ITS1 sequences of the morphs collected from same geographical area were found to be identical. The information generated in this study will help in conservation and effective breeding program to improve its productivity.

Cypermethrin Formulation (Ustad-10 EC) Induces Genotoxicity via Apoptosis, Affects Nutritional Physiology, and Modulates Immune Response in Silkworm Philosamia ricini (Lepidoptera: Saturniidae).

Cypermethrin is a pyrethroid insecticide with high insecticidal activity, low mammalian toxicity, and biodegradability. The present study aimed to determine the acute toxicity and evaluate the secondary toxic effects of a commercial formulation of cypermethrin on silkworm Philosamia ricini Hutt of Northeast India. The potential genotoxicity of cypermethrin on silkworm hemocyte was examined by comet assay, caspase activation, and annexin V affinity assay. Alteration in nutritional physiology and histoarchitecture of the gut region was evaluated. Additionally, immunotoxicological effect of cypermethrin was studied by phenoloxidase (PO), lysozyme assay, and abundance of circulating hemocytes. The LC50 value at 24-, 48-, 72-, and 96-h exposure period was recorded as 185.96, 105.34, 72.42, and 58.41 µg/liter, respectively. Approximately sevenfold increase in mean comet tail length was observed at 24 h posttreatment with sublethal concentrations of cypermethrin. Cypermethrin also induced apoptosis and activated caspase reaction in silkworm hemocytes. Moreover, a significant decrease in digestive enzyme activity was observed at higher concentrations of cypermethrin. In cypermethrin-exposed groups, alteration in histoarchitecture was also observed in the form of ruptured microvilli and thin, deformed, fused mucous layer. The PO enzyme and lysozyme enzyme activity was also altered with sublethal concentration of cypermethrin. Total hemocyte count was reduced to 10587.10, 10052.30, 9234.30, and 8842.60 per mm3 with 10, 20, 30, and 40 µg/liter, respectively. The results offer new insights into the negative consequences of very low concentrations of cypermethrin formulations on nonmulberry silkworm of Northeast India.

Larval Exposure to Chlorpyrifos Affects Nutritional Physiology and Induces Genotoxicity in Silkworm Philosamia ricini (Lepidoptera: Saturniidae).

Chlorpyrifos is a most widely used organophosphate insecticide because of its cost effectiveness and degradable nature. However, this pesticide enters and contaminates the environment either by direct application, spray drifts or crop run off and shows adverse effect on the non-targeted organisms. Philosamia ricini (eri silkworm), one of the most exploited, domesticated and commercialized non mulberry silkworm is known for mass production of eri silk. The silkworm larvae get exposed to pesticide residues on the leaves of food plants. The present study investigates the effect of commercial formulation of chlorpyrifos (Pyrifos-20 EC) on eri silkworm. Initially the LC50 value of chlorpyrifos was determined at 24-96 h and further experiments were carried out with sub lethal concentrations of the chlorpyrifos after 24 h of exposure period. The potential toxicity of chlorpyrifos was evaluated as a fuction of metabolism and nutritional physiology in 3rd, 4th, and 5th instar larvae. Alteration in histoarchitecture of 5th instar eri silkworm gut exposed to sub lethal concentration of chlorpyrifos formulation was also studied. Chlorpyrifos induced genotoxicity in silkworm hemocytes was also investigated by single cell gel electrophoresis, micronuclei assay, and apoptosis assay. Herein, LC50 values of chlorpyrifos were calculated as 3.83, 3.35, 2.68, and 2.35 mg/L at 24, 48, 72, and 96h respectively. A significant decrease in trehalose activity along with digestive enzyme activity was observed in chlorpyrifos affected groups (P < 0.05). Further, genotoxicity study revealed higher tail percentage, tail length and tail moment of the damage DNA in chlorpyrifos exposed groups (P < 0.001). Moreover, at 2.0 mg/L concentration, ~10 fold increases in tail length was observed as compared to the control. Results showed activation of caspase activity following 24 h chlorpyrifos exposure (1.5 and 2.0 mg/L) in a dose-dependent manner. Moreover, in control group less number of apoptotic cells was detected, however in both chlorpyrifos exposed groups' numbers of apoptotic cells were statistically higher (P < 0.001). Taken together, this study provides evidence that chlorpyrifos pollution might have adverse effect on overall nutritional physiology and genotoxicity of eri silkworm that could lead to reduced survivability of this economically beneficial insect.

Antioxidant Effect of Sericin in Brain and Peripheral Tissues of Oxidative Stress Induced Hypercholesterolemic Rats.

This study evaluated the antioxidant effect of crude sericin extract (CSE) from Antheraea assamensis in high cholesterol fed rats. Investigation was conducted by administering graded oral dose of 0.25 and 0.5 gm/kg body weight (b.w.)/day of CSE for a period of 28 days. Experiments were conducted in 30 rats and were divided into five groups: normal control, high cholesterol fed (HCF), HCF + 0.065 gm/kg b.w./day fenofibrate (FF), HCF + sericin 0.25 gm/kg b.w./day (LSD), and HCF + sericin 0.5 gm/kg b.w./day (HSD). In brain, heart, liver, serum, and kidney homogenates nitric oxide (NO), thiobarbituric acid reactive substances (TBARS), protein carbonyl content (PCC), superoxide dismutase, reduced glutathione (GSH) was measured. LSD treatment prevented the alterations in GSH and PCC levels in hypercholesterolemic (HyC) brain tissue homogenates of rats. CSE lowers the serum total cholesterol level in HyC rats by promoting fecal cholesterol (FC) excretion. CSE increases FC level by promoting inhibition of cholesterol absorption in intestine. The endogenous antioxidant reduced significantly and the oxidative stress marker TBARS level increases significantly in the peripheral tissue of HCF rats. However, the administration of LSD and HSD exhibited a good antioxidant activity by reducing the TBARS level and increasing the endogenous antioxidant in peripheral tissue. In addition, a histological examination revealed loss of normal liver and kidney architecture in cholesterol fed rats which were retained in sericin treated groups. The findings of this study suggested that CSE improves hypercholesterolemia in rats fed a HyC diet. Clinical relevance of this effect of CSE seems worthy of further studies.

Characterization and pathogenicity assessment of gut-associated microbes of muga silkworm Antheraea assamensis Helfer (Lepidoptera: Saturniidae).

Antheraea assamensis Helfer (muga silkworm) is an economically important endemic insect species of North Eastern Region of India. The silkworm is often susceptible to infection by pathogenic bacteria, leads to a disease commonly known as flacherie which causes 40% loss per annum to the silk industry. In this study, we have reported isolation, characterization and pathogenicity assessment of gut-associated bacteria of healthy and diseased muga silkworms. Thirty five bacterial isolates were screened from the gut of healthy and diseased silkworms by morphological observation and biochemical tests. 11 and 5 strains from healthy and diseased silkworm respectively were identified by 16S rRNA gene sequencing and analyzed. Pseudomonas aeruginosa (DRK1), Ornithinibacillus bavariensis (DRK2), Achromobacter xylosoxidans (KH3) and Staphylococcus aureus (FLG1) strains were commonly found in healthy as well as diseased larvae whereas, Bacillus thuringiensis (MK1) was found only in diseased larvae. Survivability analysis was performed with the identified strains by injection and oral administration (10(4)CFU/ml). The immune response of the silkworm against the pathogen was also studied by phenoloxidase and lysozyme enzyme activity assay, total and differential hemocyte count and phagocytic activity of hemocytes. It was observed that S. aureus, P. aeruginosa and B. thuringiensis significantly reduced the survivability of silkworm (p<0.001) hence found highly pathogenic. The lethal concentrations (LC50) values of the pathogenic strains were calculated at different time intervals (24, 48, 72 and 96h) within the range from 1.38×10(2) to 3.63×10(7)CFU/ml. The pathogenic groups demonstrated inhibition of phenoloxidase activity and decreased in total hemocyte count after 48h of infection. However, the lysozyme activity increased significantly in the pathogenic groups compared to the control (p<0.05). Granulocytes and plasmatocytes showed phagocytosis whereas; other types of cells did not show any phagocytic activity. Increasing granulocytes and plasmatocytes counts corroborates the results of phagocytic activity. The present study might be helpful in understanding the disease prognosis and colonization of bacteria causing the disease in muga silkworm.

Fiber from ramie plant (Boehmeria nivea): A novel suture biomaterial.

The quest for developing an ideal suture material prompted our interest to develop a novel suture with advantageous characters to market available ones. From natural origin only silk, cotton and linen fibers are presently available in market as non-absorbable suture biomaterials. In this study, we have developed a novel, cost-effective, and biocompatible suture biomaterial from ramie plant, Boehmeria nivea fiber. Field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and thermo gravimetric analysis (TGA) results revealed the physicochemical properties of raw and degummed ramie fiber, where the former one showed desirable characteristics for suture preparation. The braided multifilament ramie suture prepared from degummed fiber exhibited excellent tensile strength. The suture found to be biocompatible towards human erythrocytes and nontoxic to mammalian cells. The fabricated ramie suture exhibited significant antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus; which can be attributed to the inherent bacteriostatic ability of ramie plant fiber. In vivo wound closure efficacy was evaluated in adult male wister rats by suturing the superficial wound incisions. Within seven days of surgery the wound got completely healed leaving no rash and scar. The role of the ramie suture in complete wound healing was supported by the reduced levels of serum inflammatory mediators. Histopathology studies confirmed the wound healing ability of ramie suture, as rapid synthesis of collagen, connective tissue and other skin adnexal structures were observed within seven days of surgery. Tensile properties, biocompatibility and wound closure efficacy of the ramie suture were comparable with market available BMSF suture. The outcome of this study can drive tremendous possibility for the utilization of ramie plant fiber for various biomedical applications.

Potential of silk fibroin/chondrocyte constructs of muga silkworm Antheraea assamensis for cartilage tissue engineering.

Articular cartilage damage represents one of the most perplexing clinical problems of musculoskeletal therapeutics due to its limited self-repair and regenerative capabilities. In this study, 3D porous silk fibroin scaffolds derived from non-mulberry muga silkworm Antheraea assamensis were fabricated and examined for their ability to support cartilage tissue engineering. Additionally, Bombyx mori and Philosamia ricini silk fibroin scaffolds were utilized for comparative studies. Herein, the fabricated scaffolds were thoroughly characterized and compared for cartilaginous tissue formation within the silk fibroin scaffolds seeded with primary porcine chondrocytes and cultured in vitro for 2 weeks. Surface morphology and structural conformation studies revealed the highly interconnected porous structure (pore size 80-150 µm) with enhanced stability within their structure. The fabricated scaffolds demonstrated improved mechanical properties and were followed-up with sequential experiments to reveal improved thermal and degradation properties. Silk fibroin scaffolds of A. assamensis and P. ricini supported better chondrocyte attachment and proliferation as indicated by metabolic activities and fluorescence microscopic studies. Biochemical analysis demonstrated significantly higher production of sulphated glycosaminoglycans (sGAGs) and type II collagen in A. assamensis silk fibroin scaffolds followed by P. ricini and B. mori scaffolds (p < 0.001). Furthermore, histochemistry and immunohistochemical studies indicated enhanced accumulation of sGAGs and expression of collagen II. Moreover, the scaffolds in a subcutaneous model of rat demonstrated in vivo biocompatibility after 8 weeks of implantation. Taken together, these results demonstrate the positive attributes from the non-mulberry silk fibroin scaffold of A. assamensis and suggest its suitability as a promising scaffold for chondrocyte based cartilage repair.

Milled non-mulberry silk fibroin microparticles as biomaterial for biomedical applications.

Silk fibroin has been widely employed in various forms as biomaterials for biomedical applications due to its superb biocompatibility and tunable degradation and mechanical properties. Herein, silk fibroin microparticles of non-mulberry silkworm species (Antheraea assamensis, Antheraea mylitta and Philosamia ricini) were fabricated via a top-down approach using a combination of wet-milling and spray drying techniques. Microparticles of mulberry silkworm (Bombyx mori) were also utilized for comparative studies. The fabricated microparticles were physico-chemically characterized for size, stability, morphology, chemical composition and thermal properties. The silk fibroin microparticles of all species were porous (∼5µm in size) and showed nearly spherical morphology with rough surface as revealed from dynamic light scattering and microscopic studies. Non-mulberry silk microparticles maintained the typical silk-II structure with ß-sheet secondary conformation with higher thermal stability. Additionally, non-mulberry silk fibroin microparticles supported enhanced cell adhesion, spreading and viability of mouse fibroblasts than mulberry silk fibroin microparticles (p<0.001) as evidenced from fluorescence microscopy and cytotoxicity studies. Furthermore, in vitro drug release from the microparticles showed a significantly sustained release over 3 weeks. Taken together, this study demonstrates promising attributes of non-mulberry silk fibroin microparticles as a potential drug delivery vehicle/micro carrier for diverse biomedical applications.

Silk fibroin-keratin based 3D scaffolds as a dermal substitute for skin tissue engineering.

Development of highly vascular dermal tissue-engineered skin substitutes with appropriate mechanical properties and cellular cues is in need for significant advancement in the field of dermal reconstruction. Limitations have been imposed on natural biomaterials despite their superb biocompatibility hence, studies in biomaterial blending have been ongoing. Herein, we investigated blends of silk fibroin and human hair-derived keratin as wound-healing substrates that promote enhanced fibroblast cell adhesion and proliferation. Three-dimensional (3D) blended scaffolds were fabricated by freeze-drying, and their physico-chemical, mechanical and degradable properties were extensively characterized. Cytocompatibility tests observing cell adhesion and cell proliferation have shown significant enhancements in blended scaffolds. Also, its structural composition with high porosity (>85%) and interconnected pores in the range of 100-120 microns further confirms the superiority of the complex compared to its counterparts. FTIR studies identified the enhanced stability within its structure and were followed-up with sequential experiments to demonstrate improved thermal, degradation, and mechanical properties. Furthermore, immunohistochemical staining revealed greater expression of collagen type I in the cultured cells, indicating functional fibroblast proliferation and, hence, the exciting potential of this construct for dermal applications. Taken together, this study demonstrates the promising attributes from blended biomaterials and specifically present silk fibroin and human hair keratin blended scaffolds as a promising dermal substitute for skin tissue engineering.

Correction: Silk fibroin-keratin based 3D scaffolds as a dermal substitute for skin tissue engineering.

Correction for 'Silk fibroin-keratin based 3D scaffolds as a dermal substitute for skin tissue engineering' by Nandana Bhardwaj et al., Integr. Biol., 2015, DOI: 10.1039/c4ib00208c.

Tissue-engineered cartilage: the crossroads of biomaterials, cells and stimulating factors.

Damage to cartilage represents one of the most challenging tasks of musculoskeletal therapeutics due to its limited propensity for healing and regenerative capabilities. Lack of current treatments to restore cartilage tissue function has prompted research in this rapidly emerging field of tissue regeneration of functional cartilage tissue substitutes. The development of cartilaginous tissue largely depends on the combination of appropriate biomaterials, cell source, and stimulating factors. Over the years, various biomaterials have been utilized for cartilage repair, but outcomes are far from achieving native cartilage architecture and function. This highlights the need for exploration of suitable biomaterials and stimulating factors for cartilage regeneration. With these perspectives, we aim to present an overview of cartilage tissue engineering with recent progress, development, and major steps taken toward the generation of functional cartilage tissue. In this review, we have discussed the advances and problems in tissue engineering of cartilage with strong emphasis on the utilization of natural polymeric biomaterials, various cell sources, and stimulating factors such as biophysical stimuli, mechanical stimuli, dynamic culture, and growth factors used so far in cartilage regeneration. Finally, we have focused on clinical trials, recent innovations, and future prospects related to cartilage engineering.

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms.

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk-based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials.