Inventing a facile method to construct Bombyx mori (B. mori) silk fibroin nanocapsules for drug delivery

Bombyx mori (B. mori) silk fibroin (SF) microcapsules have acted as a great candidate in delivering drugs. However, it is difficult to fabricate SF nanocapsules using the present layer-by-layer (LBL) technique. In addition, the current SF microcapsules have limits in loading negatively charged drugs. Here, we invent a novel LBL method by introducing silane (APTES) as a structure indicator to produce SF nanocapsules that can load drugs with negative or positive charge. LBL assembly was completed by alternately coating SF and APTES on the template of polystyrene (PS) nanospheres by electrostatic attraction. SF nanocapsules were obtained after removal of the PS templates. Zeta potential analysis proved LBL assembly was indeed driven by the interaction between negative charge of SF and positive charge of APTES. Fluorescence images and electric microscope images indicated that SF nanocapsules had a hollow and stable structure with diameter at nearly 250 nm. The highest encapsulation rate of DOX or Ce6 were up to 80% and 90%, respectively, indicating SF nanocapsules have a high loading capability for both cationic and anionic drugs. In vitro cell experiments proved the biocompatibility of SF nanocapsules and their burst drug release in response to acidic environment. Furthermore, chemotherapy and photodynamic therapy proved SF nanocapsules loaded with DOX or Ce6 had significant inhibition on tumor cells. Our results suggested that this LBL technique is a facile method for polymers with negative charge to fabricate nanocapsules for antitumor drug carrier.

A novel LBL method was proposed here by introducing silane to produce stable SF nanocapsules for better drug delivery.     

Multiple forms of U2 snRNA coexist in the silk moth Bombyx mori

Eight U2 snRNA variants were isolated from several Bombyx mori U2-specific RT-PCR libraries. U2 sequences and secondary structures were generated and examined in terms of potential RNA and protein interactions. Analysis indicated that nucleotide changes occurred in both stem/loop and single-stranded areas. Changes in the double stranded areas were either compensatory, single substitutions (e.g. C <--> U) or prevented the double-stranded formation of one or two base pairs. The polymorphisms were clustered in moderately conserved regions. Some of the changes observed generated stronger base pairing. Inter-species conserved protein or RNA-binding sites were relatively unaffected. No polymorphic sites were found in known functional sequences. Bombyx mori and Drosophila melanogaster U2 sequences are 95% and 70% similar at the 5'- and the 3'-ends of the molecule, respectively. Phylogenetic analysis of the U2 sequences demonstrates remarkable conservation across species.     

Ice-regenerated flame retardant and robust film of Bombyx mori silk fibroin and POSS nano-cages

In this study, we present a simple method to prepare and control the structure of regenerated hybrid silkworm silk films through icing. A regenerated hybrid silk (RHS) film consisting of a micro-fibrillar structure was obtained by partially dissolving amino-functionalized polyhedral oligomeric silsesquioxanes (POSS) and silk fibers in a CaCl₂–formic acid solution. After immersion in water and icing, the obtained films of RHS showed polymorphic and strain-stiffening behaviors with mechanical properties that were better than those observed in dry or wet-regenerated silk. It was also found that POSS endowed the burning regenerated silk film with anti-dripping properties. The higher β-sheet content observed in the ice-regenerated hybrid micro-fibrils indicates a useful route to fabricate regenerated silk with physical and functional properties, i.e. strain-stiffening, similar to those observed to date in natural spider silk counterpart and synthetic rubbers, and anti-dripping of the flaming melt. Related carbon nanotube composites are considered for comparison.

In this study, we present a simple method to prepare and control the structure of regenerated hybrid silkworm silk films through icing.     

Subchronic toxicity of magnesium oxide nanoparticles to Bombyx mori silkworm

Despite many research efforts devoted to the study of the effects of magnesium oxide nanoparticles (MgO NPs) on cells or animals in recent years, data related to the potential long-term effects of this nanomaterial are still scarce. The aim of this study is to explore the subchronic effects of MgO NPs on Bombyx mori silkworm, a complete metamorphosis insect with four development stages (egg, larva, pupa, month). With this end in view, silkworm larvae were exposed to MgO NPs at different mass concentrations (1%, 2%, 3% and 4%) throughout their fifth instar larva. Their development, survival rate, cell morphology, gene expressions, and especially silk properties were compared with a control. The results demonstrate that MgO NPs have no significant negative impact on the growth or tissues. The cocooning rate and silk quality also display normal results. However, a total of 806 genes are differentially expressed in the silk gland (a vital organ for producing silk). GO (Gene Ontology) results show that the expression of many genes related to transporter activity are significantly changed, revealing that active transport is the main mechanism for the penetration of MgO NPs, which also proves that MgO NPs are adsorbed by cells. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis demonstrates that the longevity regulating pathway-worm, peroxisome and MAPK signaling pathway are closely involved in the biological effects of MgO NPs. Overall, subchronic exposure to MgO NPs induced no apparent negative impact on silkworm growth or silks but changed the expressions of some genes.

The subchronic toxicity of MgO NPs was studied by silkworm model, from the levels of animal entirety, tissues, and genes.     

Function of a TGF‐β inducible nuclear protein in the silk gland in Bombyx mori

A TGF‐β inducible nuclear protein 1 (BmTINP1) was cloned from silkworm, Bombyx mori. Polyclonal antibodies against BmTINP1 were produced and subsequently used in immunoblotting and immunohistochemistry analyses. The immunoblotting analyses demonstrated that BmTINP1 was specifically expressed in the anterior silk gland (ASG) and the middle silk gland (MSG) but not in the posterior silk gland (PSG). There were two bands that suggested the existence of an isoform of BmTINP1. The expression profiles of BmTINP1 in ASGs and MSGs were similar, and they manifested a high level of expression throughout the period during which silk gland grew exponentially. Immunohistochemistry results revealed that BmTINP1 was translocated from the nucleus into the cytoplasm when larvae developed from the 4th‐HCS into the 5th instar. 20‐hydroxyecdysone (20E) promotes the translocation, while the methoprene [a juvenile hormone (JH) analog] restrains the process. Our findings indicate that BmTINP1 is involved in silk produce along with the rapid growth of ASGs and MSGs during the last instar larvae, and the process could be regulated by hormones via control of BmTINP1 translocation from the nucleus to the cytoplasm.     

Cloning and expression analysis of takeout/JHBP family genes of silkworm, Bombyx mori

A Bombyx EST cDNA database was searched using the Drosophila takeout gene and nine cDNAs were obtained. The homology search suggested that these genes are widespread in insects and organize a large gene family, and that they have hydrophobic ligands. A phylogenetic tree indicated that the genes are first divided into two large groups, juvenile hormone binding protein and other protein genes, and the latter group diversified within a short time at an early stage. The expression study of five Bombyx genes indicated that they are expressed in various tissues and are regulated by development and feeding conditions. The Bombyx genes might have roles related to the regulation of metabolism, growth or development related to nutritional conditions.     

Studies on the chitin/chitosan binding properties of six cuticular proteins analogous to peritrophin 3 from Bombyx mori

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated‐chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3‐A1, BmCPAP3‐A2, BmCPAP3‐B, BmCPAP3‐C, BmCPAP3‐D1 and BmCPAP3‐D2) were cloned and expressed in Escherichia coli and purified using metal‐chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3‐D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3‐D1 was similar to BmCPAP3‐A1 and BmCPAP3‐C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin‐binding protein, BmCPAP3‐D1, which exhibits high binding affinity to deacetylated chitin.     

丝素/褐藻多糖硫酸酯复合膜的理化性质

背景:丝素膜α-螺旋的结构是水溶性的而且不稳定,不能直接作为医用材料,具而β-折叠构象更为稳定.目的:利用褐藻多糖硫酸酯导致丝素膜的构象转变来制备β折叠构象的丝素膜,观察膜的理化件质.设计、时间及地点:生物材料复合膜的理化性质观察实验,于2003-11/2006-08在中科院北京化学所、承德石油高等专科学校化学实验中心完成.材料:褐藻多糖硫酸酯,从海带中提取,在承德石油高等专科学校化学实验中心完成,蚕丝购于中国进出口公司.方法:将丝素溶液和褐藻多糖硫酸酯的水溶液按一定比例混合,静置脱泡后,注入聚乙烯模具内,于25℃.相对湿度65%的环境干燥成膜,当褐藻多糖硫酸酯含量≤20%时,成膜性较好.根据复合膜中褐藻多糖硫酸酯和丝素的质量比0∶100、5∶95、10∶90、20∶80、100∶0,将膜分别标记.主要观察指标:采用红外光谱、X射线衍射、热失重分析表征观察制成膜的理化性质.结果:红外光谱、X射线衍射结果表明,由于褐藻多糖硫酸酯的引入,促使了丝素β-折替构象的形成.丝素从无规线团转变为β-折叠构象归结于丝素与褐藻多糖硫酸酯之间存在着氢键的相互作用.热失重分析表明,具有β-折叠结晶结构的丝素复合膜热稳定性要比无定形丝素膜高.结论:利用褐藻多糖硫酸酯导致丝素膜的构象转变来制备β折叠构象的丝素膜,构象的转变可通过红外光谱、X射线衍射证实.具有β-折叠结晶结构的丝素膜的热稳定性要比无定形丝素膜高.     

B96Bom encodes a Bombyx mori tyramine receptor negatively coupled to adenylate cyclase

A cDNA encoding a biogenic amine receptor (B96Bom) was isolated from silkworm (Bombyx mori) larvae, and the ligand response of the receptor stably expressed in HEK-293 cells was examined. Tyramine (TA) at 0.1-100 micro m reduced forskolin (10 micro m)-stimulated intracellular cAMP levels by approximately 40%. The inhibitory effect of TA at 1 micro m was abolished by yohimbine and chlorpromazine (each 10 micro m). Although octopamine (OA) also reduced the cAMP levels, the potency was at least two orders of magnitude lower than that of TA. Furthermore, unlabelled TA (IC50 = 5.2 nm) inhibited specific [3H]TA binding to the membranes of B96Bom-transfected HEK-293 cells more potently than did OA (IC50 = 1.4 micro m) and dopamine (IC50 = 1.7 micro m). Taken together with the result of phylogenetic analysis, these findings indicate that the B96Bom receptor is a B. mori TA receptor, which is negatively coupled to adenylate cyclase. The use of this expression system should facilitate physiological studies of TA receptors as well as structure-activity studies of TA receptor ligands.     

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.     

A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm

Body shape is one of the most prominent and basic characteristics of any organism. In insects, abundant variations in body shape can be observed both within and amongst species. However, the molecular mechanism underlying body shape fine‐tuning is very complex and has been largely unknown until now. In the silkworm Bombyx mori, the tubby (tub) mutant has an abnormal short fat body shape and the abdomen of tub larvae expands to form a fusiform body shape. Morphological investigation revealed that the body length was shorter and the body width was wider than that of the Dazao strain. Thus, this mutant is a good model for studying the molecular mechanisms of body shape fine‐tuning. Using positional cloning, we identified a gene encoding the serine protease homologue, B. mori scarface (Bmscarface), which is associated with the tub phenotype. Sequence analysis revealed a specific 312‐bp deletion from an exon of Bmscarface in the tub strain. In addition, recombination was not observed between the tub and Bmscarface loci. Moreover, RNA interference of Bmscarface resulted in the tub‐like phenotype. These results indicate that Bmscarface is responsible for the tub mutant phenotype. This is the first study to report that mutation of a serine protease homologue can induce an abnormal body shape in insects.     

A novel hAT element in Bombyx mori and Rhodnius prolixus: its relationship with miniature inverted repeat transposable elements (MITEs) and horizontal transfer

Comparative analysis of transposable elements (TEs) from different species can make it possible to reconstruct their history over evolutionary time. In this study, we identified a novel hAT element in Bombyx mori and Rhodnius prolixus with characteristic GGGCGGCA repeats in its subterminal region. Meanwhile, phylogenetic analysis demonstrated that the elements in these two species might represent a separate cluster of the hAT superfamily. Strikingly, a previously identified miniature inverted repeat transposable element (MITE) shared high identity with this autonomous element across the entire length, supporting the hypothesis that MITEs are derived from the internal deletion of DNA transposons. Interestingly, identity of the consensus sequences of this novel hAT element between B. mori and R. prolixus, which diverged about 370 million years ago, was as high as 96.5% over their full length (about 3.6 kb) at the nucleotide level. The patchy distribution amongst species, coupled with overall lack of intense purifying selection acting on this element, suggest that this novel hAT element might have experienced horizontal transfer between the ancestors of B. mori and R. prolixus. Our results highlight that this novel hAT element could be used as a potential tool for germline transformation of R. prolixus to control the transmission of Trypanosoma cruzi, which causes Chagas disease.