Effects of Osiris9a on Silk Properties in Bombyx mori Determined by Transgenic Overexpression

Osiris is an insect-specific gene family with multiple biological roles in development, phenotypic polymorphism, and protection. In the silkworm, we have previously identified twenty-five Osiris genes with high evolutionary conservation and remarkable synteny among several insects. Bombxy mori Osiris9a (BmOsi9a) is expressed only in the silk gland, particularly in the middle silk gland (MSG). However, the biological function of BmOsi9a is still unknown. In this study, we overexpressed BmOsi9a in the silk gland by germline transgene expression. BmOsi9a was overexpressed not only in the MSG but also in the posterior silk gland (PSG). Interestingly, BmOsi9a could be secreted into the lumen in the MSG but not in the PSG. In the silk fiber, overexpressed BmOsi9a interacted with Sericin1 in the MSG, as confirmed by a co-immunoprecipitation assay. The overexpression of BmOsi9a altered the secondary structure and crystallinity of the silk fiber, thereby changing the mechanical properties. These results provide insight into the mechanisms underlying silk proteins secretion and silk fiber formation

CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells

The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori

Bending–Spinning Produces Silkworm and Spider Silk with Enhanced Mechanical Properties

Natural silk-spinning behavior endows freshly secreted (FS) silk with a bending angle. This phenomenon is universal among silkworms and spiders but remains unexplained. In this study, the angle of freshly secreted silkworm (FS-S) silk was revealed to be 70.23°–87.93° and that of freshly secreted spider major ampullate (FS-MA) silk to be 0°–50°. Furthermore, the mechanical properties of FS-S silk and FS-MA silk changed after bending, first increasing and then decreasing as the bending angle was increased (0°–360°). The angle corresponding to FS-S silk with the best mechanical properties is 67.5°–90°, and for FS-MA silk, it is 45°. Structural analysis showed that the β-sheet, crystallinity, and crystal size increased with bending angle and the orientation decreased with bending angle. Regular change of microstructure explains the trend for the mechanical property change with bending angle. These findings suggest that bending–spinning produces silkworm and spider silk with enhanced mechanical properties

Bending–Spinning Produces Silkworm and Spider Silk with Enhanced Mechanical Properties

Natural silk-spinning behavior endows freshly secreted (FS) silk with a bending angle. This phenomenon is universal among silkworms and spiders but remains unexplained. In this study, the angle of freshly secreted silkworm (FS-S) silk was revealed to be 70.23°–87.93° and that of freshly secreted spider major ampullate (FS-MA) silk to be 0°–50°. Furthermore, the mechanical properties of FS-S silk and FS-MA silk changed after bending, first increasing and then decreasing as the bending angle was increased (0°–360°). The angle corresponding to FS-S silk with the best mechanical properties is 67.5°–90°, and for FS-MA silk, it is 45°. Structural analysis showed that the β-sheet, crystallinity, and crystal size increased with bending angle and the orientation decreased with bending angle. Regular change of microstructure explains the trend for the mechanical property change with bending angle. These findings suggest that bending–spinning produces silkworm and spider silk with enhanced mechanical properties

Bending–Spinning Produces Silkworm and Spider Silk with Enhanced Mechanical Properties

Natural silk-spinning behavior endows freshly secreted (FS) silk with a bending angle. This phenomenon is universal among silkworms and spiders but remains unexplained. In this study, the angle of freshly secreted silkworm (FS-S) silk was revealed to be 70.23°–87.93° and that of freshly secreted spider major ampullate (FS-MA) silk to be 0°–50°. Furthermore, the mechanical properties of FS-S silk and FS-MA silk changed after bending, first increasing and then decreasing as the bending angle was increased (0°–360°). The angle corresponding to FS-S silk with the best mechanical properties is 67.5°–90°, and for FS-MA silk, it is 45°. Structural analysis showed that the β-sheet, crystallinity, and crystal size increased with bending angle and the orientation decreased with bending angle. Regular change of microstructure explains the trend for the mechanical property change with bending angle. These findings suggest that bending–spinning produces silkworm and spider silk with enhanced mechanical properties

Bending–Spinning Produces Silkworm and Spider Silk with Enhanced Mechanical Properties

Natural silk-spinning behavior endows freshly secreted (FS) silk with a bending angle. This phenomenon is universal among silkworms and spiders but remains unexplained. In this study, the angle of freshly secreted silkworm (FS-S) silk was revealed to be 70.23°–87.93° and that of freshly secreted spider major ampullate (FS-MA) silk to be 0°–50°. Furthermore, the mechanical properties of FS-S silk and FS-MA silk changed after bending, first increasing and then decreasing as the bending angle was increased (0°–360°). The angle corresponding to FS-S silk with the best mechanical properties is 67.5°–90°, and for FS-MA silk, it is 45°. Structural analysis showed that the β-sheet, crystallinity, and crystal size increased with bending angle and the orientation decreased with bending angle. Regular change of microstructure explains the trend for the mechanical property change with bending angle. These findings suggest that bending–spinning produces silkworm and spider silk with enhanced mechanical properties

Bending–Spinning Produces Silkworm and Spider Silk with Enhanced Mechanical Properties

Natural silk-spinning behavior endows freshly secreted (FS) silk with a bending angle. This phenomenon is universal among silkworms and spiders but remains unexplained. In this study, the angle of freshly secreted silkworm (FS-S) silk was revealed to be 70.23°–87.93° and that of freshly secreted spider major ampullate (FS-MA) silk to be 0°–50°. Furthermore, the mechanical properties of FS-S silk and FS-MA silk changed after bending, first increasing and then decreasing as the bending angle was increased (0°–360°). The angle corresponding to FS-S silk with the best mechanical properties is 67.5°–90°, and for FS-MA silk, it is 45°. Structural analysis showed that the β-sheet, crystallinity, and crystal size increased with bending angle and the orientation decreased with bending angle. Regular change of microstructure explains the trend for the mechanical property change with bending angle. These findings suggest that bending–spinning produces silkworm and spider silk with enhanced mechanical properties

Bending–Spinning Produces Silkworm and Spider Silk with Enhanced Mechanical Properties

Natural silk-spinning behavior endows freshly secreted (FS) silk with a bending angle. This phenomenon is universal among silkworms and spiders but remains unexplained. In this study, the angle of freshly secreted silkworm (FS-S) silk was revealed to be 70.23°–87.93° and that of freshly secreted spider major ampullate (FS-MA) silk to be 0°–50°. Furthermore, the mechanical properties of FS-S silk and FS-MA silk changed after bending, first increasing and then decreasing as the bending angle was increased (0°–360°). The angle corresponding to FS-S silk with the best mechanical properties is 67.5°–90°, and for FS-MA silk, it is 45°. Structural analysis showed that the β-sheet, crystallinity, and crystal size increased with bending angle and the orientation decreased with bending angle. Regular change of microstructure explains the trend for the mechanical property change with bending angle. These findings suggest that bending–spinning produces silkworm and spider silk with enhanced mechanical properties

Inducible LGALS3BP/90K activates antiviral innate immune responses by targeting TRAF6 and TRAF3 complex.

The galectin 3 binding protein (LGALS3BP, also known as 90K) is a ubiquitous multifunctional secreted glycoprotein originally identified in cancer progression. It remains unclear how 90K functions in innate immunity during viral infections. In this study, we found that viral infections resulted in elevated levels of 90K. Further studies demonstrated that 90K expression suppressed virus replication by inducing IFN and pro-inflammatory cytokine production. Upon investigating the mechanisms behind this event, we found that 90K functions as a scaffold/adaptor protein to interact with TRAF6, TRAF3, TAK1 and TBK1. Furthermore, 90K enhanced TRAF6 and TRAF3 ubiquitination and served as a specific ubiquitination substrate of TRAF6, leading to transcription factor NF-κB, IRF3 and IRF7 translocation from the cytoplasm to the nucleus. Conclusions: 90K is a virus-induced protein capable of binding with the TRAF6 and TRAF3 complex, leading to IFN and pro-inflammatory production