PepT1-knockout mice harbor a protective metabolome beneficial for intestinal wound healing.

Genetic knockout (KO) of peptide transporter-1 (PepT1) protein is known to provide resistance to acute colitis and colitis-associated cancer (CAC) in mouse models. However, it was unclear which molecule(s) or pathway(s) formed the basis for these protective effects. Recently, we demonstrated that the PepT1-/- microbiota is sufficient to protect against colitis and CAC. Given that PepT1 KO alters the gut microbiome and thereby changes the intestinal metabolites that are ultimately reflected in the feces, we investigated the fecal metabolites of our PepT1 KO mice. Using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted-metabolomics technique, we found that the fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. Among the altered fecal metabolites, tuberonic acid (TA) was sevenfold higher in KO mouse feces than in WT mouse feces. Accordingly, we studied whether the increased TA could direct an anti-inflammatory effect. Using in vitro models, we discovered that TA not only prevented lipopolysaccharide (LPS)-induced inflammation in macrophages but also improved the epithelial cell healing processes. Our results suggest that TA, and possibly other fecal metabolites, play a crucial role in the pathway(s) associated with the anticolitis effects of PepT1 KO.NEW & NOTEWORTHY Fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. One fecal metabolite, tuberonic acid (TA), was sevenfold higher in KO mouse feces than in WT mouse feces. TA prevented lipopolysaccharide (LPS)-induced inflammation in macrophages and improved the epithelial cell healing process.

Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system.

The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons. The mechanism by which the fibroin protein is efficiently synthesized and precisely regulated is an important aspect that has yet to be fully elucidated. Here, we describe the regulatory characteristics of the promoters of fibroin protein-encoding genes, namely, fibroin heavy chain (fibH) and fibroin light chain (fibL), using an optimized Gal4/UAS binary system. We found that (1) UAS-linked enhanced green fluorescent protein (EGFP) was effectively activated in the PSGs of Gal4/UAS transgenic silkworms, and fluorescence was continuously detected in the PSGs after complete formation of silk glands. (2) In the PSGs of fourth- and fifth-instar larvae of transgenic silkworms driven by fibL-Gal4 (LG4) or fibH-Gal4 (HG4), EGFP mRNA was detected in only day-3 to day-6 fifth-instar larvae, while the EGFP protein could be detected at each day of both larval stages. (3) High-level expression of Gal4 and UAS-linked EGFP caused a delay in PSG degradation in Gal4/UAS transgenic silkworms. (4) At the early pupal stage, EGFP fluorescence was also detected in fat bodies of Gal4/UAS transgenic silkworms, indicating that the PSG-specific EGFP was transported into fat bodies during PSG degeneration; however, the underlying mechanism needs to be further elucidated. This study provides a modified Gal4/UAS system used for efficient tissue-specific expression of target genes in the PSGs of silkworms and provides new insights into the regulatory characteristics of the promoters of key fibroin protein-encoding genes.

Ectopic Expression of Mulberry G-Proteins Alters Drought and Salt Stress Tolerance in Tobacco.

Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) play key roles in responses to various abiotic stress responses and tolerance in plants. However, the detailed mechanisms behind these roles remain unclear. Mulberry (Morus alba L.) can adapt to adverse abiotic stress conditions; however, little is known regarding the associated molecular mechanisms. In this study, mulberry G-protein genes, MaGα, MaGß, MaGγ1, and MaGγ2, were independently transformed into tobacco, and the transgenic plants were used for resistance identification experiments. The ectopic expression of MaGα in tobacco decreased the tolerance to drought and salt stresses, while the overexpression of MaGß, MaGγ1, and MaGγ2 increased the tolerance. Further analysis showed that mulberry G-proteins may regulate drought and salt tolerances by modulating reactive oxygen species' detoxification. This study revealed the roles of each mulberry G-protein subunit in abiotic stress tolerance and advances our knowledge of the molecular mechanisms underlying G-proteins' regulation of plant abiotic stress tolerance.

Plant G-protein ß subunits positively regulate drought tolerance by elevating detoxification of ROS.

Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) consist of α, ß and γ subunits and play important roles in response and tolerance to abiotic stresses in plants, but the function of the heterotrimeric G-protein ß subunit in response to drought remains unclear. In the present study, the AGB1 mutants (agb1-2-1 and agb1-3-2) were more sensitive to drought than the wild-type. The overexpression of mulberry (Morus alba L.) G-protein ß subunit in transgenic tobacco (Nicotiana tabacum L.) significantly enhanced the plants' drought tolerance. The transgenic tobacco plants had higher proline contents and peroxidase activities, and lower malonaldehyde and hydrogen peroxide contents and superoxide free radical accumulations under drought conditions. Additionally, transcript levels of the tobacco antioxidative genes, NtSOD and NtCAT, increased in drought-stressed transgenic tobacco plants. Thus, the heterotrimeric G-protein ß subunits positively regulate drought tolerance in plants.

Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system.

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.

Crohn's Disease and Ulcerative Colitis: From Pathophysiology to Novel Therapeutic Approaches.

Inflammatory bowel disease (IBD) is a non-specific autoimmune condition impacting the gastrointestinal tract, encompassing Crohn's disease (CD) and ulcerative colitis (UC) [...].

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Robust reactive oxygen species modulator hitchhiking yeast microcapsules for colitis alleviation by trilogically intestinal microenvironment renovation.

Ulcerative colitis (UC) is characterized by chronic inflammatory processes of the intestinal tract of unknown origin. Current treatments lack understanding on how to effectively alleviate oxidative stress, relieve inflammation, as well as modulate gut microbiota for maintaining intestinal homeostasis synchronously. In this study, a novel drug delivery system based on a metal polyphenol network (MPN) was constructed via metal coordination between epigallocatechin gallate (EGCG) and Fe3+. Curcumin (Cur), an active polyphenolic compound, with distinguished anti-inflammatory activity was assembled and encapsulated into MPN to generate Cur-MPN. The obtained Cur-MPN could serve as a robust reactive oxygen species modulator by efficiently scavenging superoxide radical (O2•-) as well as hydroxyl radical (·OH). By hitchhiking yeast microcapsule (YM), Cur-MPN was then encapsulated into YM to obtain CM@YM. Our findings demonstrated that CM@YM was able to protect Cur-MPN to withstand the harsh gastrointestinal environment and enhance the targeting and retention abilities of the inflamed colon. When administered orally, CM@YM could alleviate DSS-induced colitis with protective and therapeutic effects by scavenging ROS, reducing pro-inflammatory cytokines, and regulating the polarization of macrophages to M1, thus restoring barrier function and maintaining intestinal homeostasis. Importantly, CM@YM also modulated the gut microbiome to a favorable state by improving bacterial diversity and transforming the compositional structure to an anti-inflammatory phenotype as well as increasing the content of short-chain fatty acids (SCFA) (such as acetic acid, propionic acid, and butyric acid). Collectively, with excellent biocompatibility, our findings indicate that synergistically regulating intestinal microenvironment will be a promising approach for UC.

Prevention of Colitis-Associated Cancer via Oral Administration of M13-Loaded Lipid Nanoparticles.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease, is known to increase the risk of colitis-associated cancer (CAC). CAC has been found to be unresponsive to standard chemotherapy regimens, and the current treatments do not utilize effective small-molecule drugs and colon-targeted delivery systems. Previous studies indicated that the M13-nano-liposome (NL) formulation can effectively target the colon and reshape the gut microbiota in ex vivo cultures, generating altered microbial metabolites that can efficiently prevent chronic UC. In this study, we tested the cancer cell uptake ability of the NL formulation and investigated the potential of the M13-NL formulation to prevent CAC in the azoxymethane (AOM)-exposed IL10-/- mouse model. Our findings demonstrate that oral administration of M13-NL prevents tumor development in AOM-exposed IL10-/- mice, suggesting that M13-NL is a promising oral drug formulation for preventing CAC.

Oral administration of M13-loaded nanoliposomes is safe and effective to treat colitis-associated cancer in mice.

OBJECTIVE: Colitis-associated cancer (CAC) treatment lacks effective small-molecule drugs and efficient targeted delivery systems. Here, we loaded M13 (an anti-cancer drug candidate) to colon-targeting ginger-derived nanoliposomes (NL) and investigated if orally administered M13-NL could enhance the anticancer effects of M13 in CAC mouse models. METHODS: The biopharmaceutical properties of M13 were assessed by physicochemical characterizations. The in vitro immunotoxicity of M13 was assessed against PBMCs using FACS and the mutagenic potential of M13 was evaluated by the Ames assay. The in vitro efficacy of M13 was tested in 2D- and 3D-cultured cancerous intestinal cells. AOM/DSS-induced CAC mice were used to evaluate the therapeutic effects of free M13 or M13-NL on CAC in vivo. RESULTS: M13 has beneficial physiochemical properties, including high stability, and no apparent immunotoxicity or mutagenic potential in vitro. M13 is effective against the growth of 2D- and 3D-cultured cancerous intestinal cells in vitro. The in vivo safety and efficacy of M13 were significantly improved by using NL for drug delivery (p < 0.001). Oral administration of M13-NL exhibited excellent therapeutic effects in AOM/DSS-induced CAC mice. CONCLUSION: M13-NL is a promising oral drug formulation for CAC treatment.

[Progress in Cre/lox site-specific recombination system in higher eukaryotes].

Cre/lox system derived from P1 bacteriaphage can quickly and effectively achieve gene insertion, deletion, replacement, and inversion by means of site-specific recombination. As one of the most important tools for gene targeting at present, Cre/lox system has been widely used in Arabidopsis thaliana, Oryza sativa L., Mus musculus, Drosophila melanogaster, Danio rerio, and other higher eukaryotic organisms. This review roundly described the basic profile of Cre/lox system, and its application in higher eukaryotes. In addition, we also discussed the main problems and developmental trend of the Cre/lox system in this review, which can be a good reference for using Cre/lox system to realize the gene manipulations of the different high eukaryotic organisms.

Oral delivery of IL-22 mRNA-loaded lipid nanoparticles targeting the injured intestinal mucosa: A novel therapeutic solution to treat ulcerative colitis.

Oral mRNA delivery is a promising yet understudied approach for treating inflammatory bowel disease (IBD). Inspired by the colon-targeting ability of ginger-derived nanoparticles (GDNPs), we reversely engineered lipid nanoparticles that comprise the three major lipids identified in GDNPs. When mixed at the ratio found in GDNPs, the selected lipids (phosphatidic acid, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol; 5:2:3) self-assembled into new lipid nanoparticles (nLNPs) in phosphate-buffered saline. We encapsulated IL-22-mRNA within the nLNPs, as enhanced IL-22 expression in the colon is known to have potent anti-inflammatory efficacy against ulcerative colitis (UC). The IL-22 mRNA-loaded nLNPs (IL-22/nLNPs) were found to be about 200 nm in diameter and have a zeta potential of -18 mV. Oral delivery of IL-22/nLNPs elevated the protein expression level of IL-22 in the colonic mucosa of mice. In a mouse model of acute colitis, mice fed with IL-22/nLNPs experienced an accelerated healing process, as indicated by the recovery of more body weight and colon length as well as reduction of the histological index, colonic MPO activity, fecal lipocalin concentration, and mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß). Our results suggest that our reversely engineered nLNPs is an excellent mRNA delivery platform for treating ulcerative colitis.

Prevention of Ulcerative Colitis by Autologous Metabolite Transfer from Colitogenic Microbiota Treated with Lipid Nanoparticles Encapsulating an Anti-Inflammatory Drug Candidate.

Modulating the gut microbiota composition is a potent approach to treat various chronic diseases, including obesity, metabolic syndrome, and ulcerative colitis (UC). However, the current methods, such as fecal microbiota transplantation, carry a risk of serious infections due to the transmission of multi-drug-resistant organisms. Here, we developed an organism-free strategy in which the gut microbiota is modulated ex vivo and microbiota-secreted metabolites are transferred back to the host. Using feces collected from the interleukin-10 (IL-10) knockout mouse model of chronic UC, we found that a drug candidate (M13)-loaded natural-lipid nanoparticle (M13/nLNP) modified the composition of the ex vivo-cultured inflamed gut microbiota and its secreted metabolites. Principal coordinate analysis (PCoA) showed that M13/nLNP shifted the inflamed microbiota composition toward the non-inflamed direction. This compositional modification induced significant changes in the chemical profiles of secreted metabolites, which proved to be anti-inflammatory against in vitro-cultured NF-κß reporter cells. Further, when these metabolites were orally administered to mice, they established strong protection against the formation of chronic inflammation. Our study demonstrates that ex vivo modulation of microbiota using M13/nLNP effectively reshaped the microbial secreted metabolites and that oral transfer of these metabolites might be an effective and safe therapeutic approach for preventing chronic UC.

Very Early Corona Treatment-Mediated Artificial Incubation of Silkworm Eggs and Germline Transformation of Diapause Silkworm Strains.

Diapause is an important biological characteristic for many insect species to adapt to adverse environmental conditions and maintain the continuity of the race. Compared with the traditional hydrochloric acid or/and cold storage treatment methods, the artificial corona incubation technology of silkworm (Bombyx mori) eggs has many advantages including, the absence of pollution, easy operation and safety. However, this technology has not yet been applied in sericulture. In this study, we developed a novel artificial corona instrument to successfully disrupt the diapause of newly laid and refrigerated eggs from various Chinese and Japanese lineage silkworm strains. Subsequently, we invented a very early corona treatment (VECT) strategy to prevent the diapause of newly laid silkworm eggs within 4 h of oviposition. The hatching rates of the larvae were more than 95% in all diapause silkworm strains, which was comparable to the effect of the traditional HCl treatment strategy. In addition, we developed a combination strategy of VECT and pre-blastoderm microinjection and successfully created transgenic silkworms in various diapause strains. The results of the current study can aid in improving the corona artificial incubation technology and promote its application in sericulture.

Atomic Force Microscopy to Characterize Ginger Lipid-Derived Nanoparticles (GLDNP).

We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols). To construct a nanocarrier that is more clearly defined than GDNP-2, we isolated lipids from GDNP-2 and demonstrated that they could self-assemble into ginger lipid-derived nanoparticles (GLDNP) in an aqueous solution. GLDNP can be used as a nanocarrier to deliver drug candidates such as 6-shogaol or its metabolites (M2 and M13) to the colon. To characterize the nanostructure of GLDNP, our lab extensively used atomic force microscopy (AFM) technique as a tool for visualizing the morphology of the drug-loaded GLDNP. Herein, we provide a detailed protocol for demonstrating such a process.

Orally Administered Natural Lipid Nanoparticle-Loaded 6-Shogaol Shapes the Anti-Inflammatory Microbiota and Metabolome.

The past decade has seen increasing interest in microbiota-targeting therapeutic strategies that aim to modulate the gut microbiota's composition and/or function to treat chronic diseases, such as inflammatory bowel disease (IBD), metabolic symptoms, and obesity. While targeting the gut microbiota is an innovative means for treating IBD, it typically requires an extended treatment time, hampering its potential application. Herein, using an established natural-lipid nanoparticle (nLNP) platform, we demonstrate that nLNPs encapsulated with the drug candidate 6-shogaol (6S/nLNP) distinctly altered microbiota composition within one day of treatment, significantly accelerating a process that usually requires five days using free 6-shogaol (6S). In addition, the change in the composition of the microbiota induced by five-day treatment with 6S/nLNP was maintained for at least 15 days (from day five to day 20). The consequent alteration in the fecal metabolic profile stemming from this compositional change manifested as functional changes that enhanced the in vitro anti-inflammatory and wound-healing efficacy of macrophage cells (Raw 264.7) and epithelial cells (Caco-2 BBE1), respectively. Further, this metabolic compositional change, as reflected in an altered metabolic profile, promoted a robust anti-inflammatory effect in a DSS-induced mouse model of acute colitis. Our study demonstrates that, by near-instantly modulating microbiota composition and function, an nLNP-based drug-delivery platform might be a powerful tool for treating ulcerative colitis.

The C-terminal tail of the plant endosomal-type NHXs plays a key role in its function and stability.

Typically, Na+/H+ antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K+ and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters.

Electrophoretic deposition of silk fibroin coatings with pre-defined architecture to facilitate precise control over drug delivery.

The therapeutic precision and clinical applicability of drug-eluting coatings can be substantially improved by facilitating tunable drug delivery. However, the design of coatings which allows for precise control over drug release kinetics is still a major challenge. Here, a double-layered silk fibroin (SF) coating system was constructed by sequential electrophoretic deposition. A mixture of dissolved Bombyx mori SF (bmSF) molecules and pre-made bmSF nanospheres at different ratios was deposited as under-layer. Subsequently, this underlayer was covered by a top-layer comprising Antheraea pernyi SF (apSF) molecules (rich in arginylglycylaspartic acid, RGD) to improve the cellular response of the resulting double-layered coatings. Additionally, model drug doxycycline was either pre-mixed with dissolved bmSF molecules or pre-loaded into pre-made bmSF nanospheres at the same amount before their mixing and deposition. The thickness and nanosphere content of the under-layer architecture were proportional to the deposition time and nanosphere concentration in precursor mixtures, respectively. The surface topography, wettability, degradation rate and adhesion strength were comparable within the double-layered coating system. As expected, RGD-rich apSF top-layer improved cell adhesion, spreading and proliferation compared with bmSF top-layer. Furthermore, the amount and duration of drug release increased linearly with increasing nanosphere concentration at fixed deposition time, whereas drug release amount increased linearly with increasing deposition time. These results indicate that the dosage and kinetics of loaded drugs can be quantitatively tailored by altering nanosphere concentration and deposition time as main processing parameters. Overall, this study illustrates the strong potential of pre-defining coating architecture to facilitate control over drug delivery.

Tumor Microenvironment-Responsive Nanococktails for Synergistic Enhancement of Cancer Treatment via Cascade Reactions.

A combination treatment strategy that relies on the synergetic effects of different therapeutic approaches has been considered to be an effective method for cancer therapy. Herein, a chemotherapeutic drug (doxorubicin, Dox) and a manganese ion (Mn2+) were co-loaded into regenerated silk fibroin-based nanoparticles (NPs), followed by the surface conjugation of phycocyanin (PC) to construct tumor microenvironment-activated nanococktails. The resultant PC-Mn@Dox-NPs showed increased drug release rates by responding to various stimulating factors (acidic pH, hydrogen peroxide (H2O2), and glutathione), revealing that they could efficiently release the payloads (Dox and Mn2+) in tumor cells. The released Dox could not only inhibit the growth of tumor cells but also generated a large amount of H2O2. The elevated H2O2 was decomposed into the highly harmful hydroxyl radicals and oxygen through an Mn2+-mediated Fenton-like reaction. Furthermore, the generated oxygen participated in photodynamic therapy (PDT) and produced abundant singlet oxygen. Our investigations demonstrate that these PC-Mn@Dox-NPs exhibit multiple bioresponsibilities and favorable biosafety. By integrating Dox-induced chemotherapy, Mn2+-mediated chemodynamic therapy, and PC-based PDT via cascade reactions, PC-Mn@Dox-NPs achieved enhanced in vitro and in vivo anticancer efficacies compared to all the mono- or dual-therapeutic approaches. These findings reveal that PC-Mn@Dox-NPs can be exploited as a promising nanococktail for cascade reaction-mediated synergistic cancer treatment.

New insight into the mechanism of in vivo fibroin self-assembly and secretion in the silkworm, Bombyx mori.

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.