Search Results (49)

Anterior cruciate ligament (ACL) rupture causes joint instability and increases the risk of osteoarthritis due to the ligament’s limited healing capacity. Silk, particularly from Bombyx mori, combines high cytocompatibility with robust biomechanical properties. Its main components are fibroin and sericin, with the latter usually being removed to reduce immunogenicity and improve biocompatibility. Silk threads were processed either as raw silk (designated as “untreated”) or subjected to a patented degumming procedure (DE102021118652A1) to obtain purified silk. Both variants were used alone or in combination with poly(L-lactic acid-co-caprolactone) (P(LA-CL)) fibers, yielding four scaffold groups: untreated silk, purified silk, untreated silk/P(LA-CL), and purified silk/P(LA-CL). Three-layer scaffolds were fabricated using a zigzag embroidery pattern. Structural analysis revealed scaffold porosity of ≈38% for silk, ≈46% for purified silk, and up to ≈70% for scaffolds containing P(LA-CL). Uniaxial tensile testing showed that purified silk scaffolds achieved the highest maximum force at break (≈684 N), whereas elongation at maximum force was limited in the hybrid scaffolds—silk/P(LA-CL) ≈ 28% and p-silk/P(LA-CL) ≈ 32%—despite the high intrinsic extensibility of P(LA-CL). All scaffolds supported cell adhesion and showed no cytotoxicity. P-silk and p-silk/P(LA-CL) scaffolds exhibited the highest fibroblast adherence and pronounced paxillin expression, indicating strong cell–material interactions. Gene expression of ligament-related ECM components and connexin 43 was maintained across all groups. These results demonstrate that embroidered silk fibroin scaffolds provide a reproducible architecture with tunable porosity and mechanical properties, supporting fibroblast colonization and ligament-specific ECM expression. Such scaffolds represent promising candidates for ACL tissue engineering and future graft development. Full article

Background/Objectives: The larval stage plays a pivotal role in determining caste and sex in Apis mellifera. This study integrates RNA-seq, WGCNA, and alternative splicing analyses to explore gene expression differences among 2-day-old worker, drone, and queen larvae. Methods: RNA-seq was conducted on 2-day-old larvae from all three castes. Differential expression, WGCNA, and alternative splicing patterns were investigated. A deep learning TCN model was trained using WGCNA-derived modules and demonstrated high classification accuracy. Results: The TCN model highlighted a top-10 gene set, including PDHB, Fibroin3, and LOC724161. Significant caste- and sex-specific splicing events were detected in Tk, Csd, and Fem, with AF events being most prevalent. Splicing differences between sexes exceeded those observed among castes. Conclusions: The 2-day-old larval stage is crucial for both caste and sex differentiation in honeybees. This study identifies key genes and splicing events, offering new insights into the molecular mechanisms underlying caste formation and sex determination. Full article

LIM homeodomain (LIM-HD) is a versatile family of transcription factors that act as master regulators in various developmental processes of eukaryotes, and one of the LIM-HD encoded genes is the arrowhead (AWH). In silkworm Bombyx mori, the Arrowhead gene (BmAWH) functions as a key component activating all three fibroin genes in the silk glands of B. mori, but the potential pleiotropic effects of BmAWH on various tissues of the silkworm is yet to be discovered. The objective of this study is to investigate the functional role of a BmAWH gene in the B. mori (Dazao) developmental process, using the piggyBac-based transgene technique. The size of transgenic line silk glands have become smaller, resulting in the reduction in whole cocoon weight, cocoon shell weight, and cocoon–shell ratio. Overexpression of BmAWH has induced significant changes in juvenile hormone levels in female larvae at the fifth instar larval stage. Female reproductive defects (reduction in fecundity rate, abnormal egg morphology) were observed. In addition, transgenic line larvae exhibit the complete disappearance of larval body patterns and color (melanin pigmentation). Since the LIM-HD protein functions to orchestrate complex developmental programs, this study may shed light on evolutionary adaptations and the divergence of insect gene functions. Full article

Phoxim is a pesticide extensively applied in mulberry fields, and residues may persist on leaves even after the recommended pre-harvest interval. However, the potential risks of these residues to Bombyx mori L. (Lepidoptera: Bombycidae) have long been overlooked. The results demonstrated that chronic low-dose exposure from the second to fifth instars significantly impaired silkworm development and silk production. Specifically, larvae in the 0.316 μg/mL treatment group (1/2 LC₅₀) exhibited a significant reduction in body weight, while the cocoon shell ratio was significantly decreased in both the 0.079 μg/mL (1/8 LC₅₀) and 1/2 LC₅₀ groups. Cocoon deformities were observed in the 0.032 μg/mL (1/20 LC₅₀), 1/8 LC₅₀, and 1/2 LC₅₀ groups. Histopathological analysis revealed silk gland damage in the treatment groups, with severity increasing with higher phoxim concentrations. Biochemical analyses indicated elevated malondialdehyde (MDA) levels accompanied by increased superoxide dismutase (SOD) and peroxidase (POD) activities. Notably, phoxim exposure selectively reduced juvenile hormone (JH) titers without affecting ecdysone titers. JH-regulated genes including the receptors Met1 and Met2, and transcription factors Kr-h1 and Dimm were downregulated, accompanied by suppressed expression of the fibroin synthesis gene Fib-H. These results collectively indicate that chronic low-concentration phoxim exposure disrupts endocrine regulation, damages silk gland integrity, and ultimately reduces silk production in silkworm. Full article

The transcription factor erythroid 2-related factor 2 (Nrf2) is a central regulator of cellular defense mechanisms against oxidative stress and inflammation. Keap1 (Kelch-like ECH-associated protein 1) regulates Nrf2 activity by ubiquitination-mediated cytoplasmic retention, thereby suppressing its nuclear translocation and subsequent transcriptional activation of genes encoding phase II detoxifying enzymes. Using a structure-based virtual screening approach, we screened ~16,000 natural compounds to identify Keap1-Nrf2 PPI inhibitors. Nine compounds were identified based on their high binding affinities and favorable interactions with Keap1, primarily through non-covalent interactions. To validate the binding stability of these inhibitors, molecular dynamics (MD) simulations were performed, confirming the robustness of the Keap1–inhibitor complexes over time. Subsequent in vitro assays on human epithelial keratinocyte cells (HaCaT) revealed that six of these compounds notably upregulated Nrf2 mRNA expression, regis tering increases from 23% to 50% in comparison to the control. Notably, chebulinic acid emerged as the most potent compound, demonstrating the greatest elevation in Nrf2 expression. Penetration studies further showed that chebulinic acid, when encapsulated in silk fibroin, achieved a 0.14% penetration rate after 24 h though it could not penetrate into the stratum corneum alone. This result highlighted the potential of chebulinic acid in the use of anti-aging skincare formulations. Collectively, our findings affirmed that molecular docking is a reliable and effective approach for the identification of novel anti-aging agents targeting the Keap1-Nrf2 pathway. Full article

The steroid hormone 20-hydroxyecdysone (20E), which is known to regulate insect molting and metamorphosis, is crucial for the normal development of silk glands (SGs) in the silkworm Bombyx mori. However, how the 20E signaling pathway and its core members function in the SG remains largely unclear. Here, we report that the orphan nuclear receptor BmHR3, a 20E-response factor, plays an essential role in regulating SG development and silk protein synthesis. First, we showed that tissue-specific BmHR3 overexpression and knockout result in severe developmental defects in posterior silk glands (PSGs). Second, we revealed that BmHR3 dysfunction in PSGs dramatically represses the transcription of silk fibroin protein-coding genes, thereby inhibiting fibroin protein synthesis. Finally, we confirmed that BmHR3 can regulate fibroin protein-coding gene expression via direct and indirect mechanisms. This study elucidates the vital function of BmHR3 in B. mori SG and provides valuable information for thoroughly understanding the regulatory roles of 20E signaling in specialized insect organs. Full article

The diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae), is a serious agricultural pest that utilizes silk as a defensive mechanism, with silk fibroins playing a pivotal role in this process. Through comprehensive transcriptomic analyses, we identified 3452 differentially expressed genes (DEGs) co-expressed in the silk gland of P. xylostella and associated with silk production. The Gene Ontology (GO) analysis revealed enrichment in categories related to protein synthesis, secretion, and extracellular matrix organization, while Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis linked these genes to amino acid metabolism and protein processing pathways. Additionally, we identified three key silk fibroin genes: silk fibroin heavy chain (FibH), silk fibroin light chain (FibL), and fibrohexamerin (P25). We characterized the structure of these genes and analyzed the phylogenetic relationships, amino acid composition, hydrophilicity, and other physicochemical properties of the encoded silk fibroin proteins. The expression profiles revealed peak expression levels of these genes in the silk glands of fourth instar larvae. This integrative study enhances our understanding of the molecular mechanisms underlying silk production in P. xylostella and provides a foundation for future research into the biological roles, evolutionary trajectories, and potential applications of these silk fibroin genes in agricultural pest management and biotechnology. Full article

Ascorbic acid (AA) plays a crucial role in both the proliferation and chondrogenic differentiation potential of mesenchymal stem/medicinal signalling cells (MSCs); these are both key aspects of their general therapeutic use and their increasing use in veterinary medicine. Current immunomodulatory therapies require efficient expansion of MSCs in the laboratory, while emerging tissue regeneration strategies, such as cartilage or bone repair, aim to use differentiated MSCs and modulate the expression of chondrogenic and hypertrophic markers. Our aim was to investigate whether the addition of AA to the growth medium enhances the proliferation of canine adipose-derived MSCs (cAMSCs) grown on standard plastic surfaces and whether it affects chondrogenic differentiation potential on silk fibroin (SF) films. We assessed cell viability with trypan blue and proliferation potential by calculating population doubling. Chondrogenic induction on SF films was assessed by Alcian blue staining and gene expression analysis of chondrogenic and hypertrophic genes. The results showed that growth medium with AA significantly enhanced the proliferation of cAMSCs without affecting cell viability and modulated the expression of chondrogenic and hypertrophic genes of cAMSCs grown on SF films. Our results suggest that AA may be used in growth medium for expansion of cAMSCs and, at the same time, provide the basis for future studies to investigate the role of AA and SF in chondrogenic differentiation of MSCs. Full article

The development of a hydrogel material with a modified chemical structure of poly(vinyl alcohol) (PVA) and silk fibroin (SF) using glycidyl methacrylate (GMA) (denoted as PVA-g-GMA and SF-g-GMA) is an innovative approach in the field of biomaterials and meniscus tissue engineering in this study. The PVA-g-GMA/SF-g-GMA hydrogel was fabricated using different ratios of PVA-g-GMA to SF-g-GMA: 100/0, 75/25, 50/50, 25/75, and 0/100 (w/w of dry substances), using lithium phenyl (2,4,6-trimethylbenzoyl)phosphinate (LAP) as a free radical photoinitiator, for 10 min at a low ultraviolet (UV) intensity (365 nm, 6 mW/cm²). The mechanical properties, morphology, pore size, and biodegradability of the PVA-g-GMA/SF-g-GMA hydrogel were investigated. Finally, for clinical application, human chondrocyte cell lines (HCPCs) were mixed into PVA-g-GMA/SF-g-GMA solutions and fabricated into hydrogel to study the viability of live and dead cells and gene expression. The results indicate that as the SF-g-GMA content increased, the compressive modulus of the PVA-g-GMA/SF-g-GMA hydrogel dropped from approximately 173 to 11 kPa. The degradation rates of PVA-g-GMA/SF-g-GMA 100/0, 75/25, and 50/50 reached up to 15.61%, 17.23%, and 18.93% in 4 months, respectively. In all PVA-g-GMA/SF-g-GMA conditions on day 7, chondrocyte cell vitality exceeded 80%. The PVA-g-GMA/SF-g-GMA 75:25 and 50:50 hydrogels hold promise as a biomimetic biphasic injectable hydrogel for encapsulated augmentation, offering advantages in terms of rapid photocurability, tunable mechanical properties, favorable biological responses, and controlled degradation. Full article

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR–Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR–Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy. Full article

Silk has a long history as an exclusive textile, but also as a suture thread in medicine; nowadays, diverse cell carriers are manufactured from silk. Its advantages are manifold, including high biocompatibility, biomechanical strength and processability (approved for nearly all manufacturing techniques). Silk’s limitations, such as scarcity and batch to batch variations, are overcome by gene technology, which allows for the upscaled production of recombinant “designed” silk proteins. For processing thin fibroin filaments, the sericin component is generally removed (degumming). In contrast to many synthetic biomaterials, fibroin allows for superior cell adherence and growth. In addition, silk grafts demonstrate superior mechanical performance and long-term stability, making them attractive for anterior cruciate ligament (ACL) tissue engineering. Looking at these promising properties, this review focusses on the responses of cell types to silk variants, as well as their biomechanical properties, which are relevant for ACL tissue engineering. Meanwhile, sericin has also attracted increasing interest and has been proposed as a bioactive biomaterial with antimicrobial properties. But so far, fibroin was exclusively used for experimental ACL tissue engineering approaches, and fibroin from spider silk also seems not to have been applied. To improve the bone integration of ACL grafts, silk scaffolds with osteogenic functionalization, silk-based tunnel fillers and interference screws have been developed. Nevertheless, signaling pathways stimulated by silk components remain barely elucidated, but need to be considered during the development of optimized silk cell carriers for ACL tissue engineering. Full article

The efficient production of silkworm silk is crucial to the silk industry. Silk protein synthesis is regulated by the juvenile hormone (JH) and 20-Hydroxyecdysone (20E). Therefore, the genetic regulation of silk production is a priority. JH binding protein (JHBP) transports JH from the hemolymph to target organs and cells and protects it. In a previous study, we identified 41 genes containing a JHBP domain in the Bombyx mori genome. Only one JHBP gene, BmJHBPd2, is highly expressed in the posterior silk gland (PSG), and its function remains unknown. In the present study, we investigated the expression levels of BmJHBPd2 and the major silk protein genes in the high-silk-producing practical strain 872 (S872) and the low-silk-producing local strain Dazao. We found that BmJHBPd2 was more highly expressed in S872 than in the Dazao strain, which is consistent with the expression pattern of fibroin genes. A subcellular localization assay indicated that BmJHBPd2 is located in the cytoplasm. In vitro hormone induction experiments showed that BmJHBPd2 was upregulated by juvenile hormone analogue (JHA) treatment. BmKr-h1 upregulation was significantly inhibited by the overexpression of BmJHBPd2 (BmJHBPd2OE) at the cell level when induced by JHA. However, overexpression of BmJHBPd2 in the PSG by transgenic methods led to the inhibition of silk fibroin gene expression, resulting in a reduction in silk yield. Further investigation showed that in the transgenic BmJHBPd2OE silkworm, the key transcription factor of the JH signaling pathway, Krüppel homolog 1 (Kr-h1), was inhibited, and 20E signaling pathway genes, such as broad complex (Brc), E74A, and ultraspiracle protein (USP), were upregulated. Our results indicate that BmJHBPd2 plays an important role in the JH signaling pathway and is important for silk protein synthesis. Furthermore, our findings help to elucidate the mechanisms by which JH regulates silk protein synthesis. Full article

Ultrabithorax (Ubx) is a member of the Hox gene group involved in cell fate decisions, cell proliferation and organ identity. Its function has been extensively researched in Drosophila melanogaster but little is known about it in Lepidoptera. To uncover the function of Ubx in the development of lepidopterans, we constructed the Ubx overexpression (Ubx^OE) strain based on the Nistari strain of Bombyx mori. The Ubx^OE strain showed a small body size, transparent intersegmental membrane and abnormal posterior silk gland (PSG). In the current study, we focused on the effect of Ubx overexpression on the posterior silk gland. As the major protein product of PSG, the mRNA expression of fibroin heavy chain (Fib-H) and fibroin light chain (Fib-L) was upregulated three times in Ubx^OE, but the protein expression of Fib-H and Fib-L was not significantly different. We speculated that the overexpression of Ubx downregulated the expression of Myc and further caused abnormal synthesis of the spliceosome and ribosome. Abnormalities of the spliceosome and ribosome affected the synthesis of protein in the PSG and changed its morphology. Full article

This work describes the design, preparation, and deep investigation of “intelligent nanobiomaterials” that fulfill the safety rules and aim to serve as “signal deliverers” for osteogenesis, harboring a specific peptide that promotes and enhances osteogenesis at the end of their hydrogel fibers. The de novo synthesized protein fibers, besides their mechanical properties owed to their protein constituents from elastin, silk fibroin and mussel-foot adhesive protein-1 as well as to cell-attachment peptides from extracellular matrix glycoproteins, incorporate the Bone Morphogenetic Protein-2 (BMP2) peptide (AISMLYLDEN) that, according to our studies, serves as “signal deliverer” for osteogenesis. The osteogenetic capacity of the biomaterial has been evidenced by investigating the osteogenic marker genes ALP, RUNX2, Osteocalcin, COL1A1, BMPR1A, and BMPR2, which were increased drastically in cells cultured on scaffold-BMP2 for 21 days, even in the absence of osteogenesis medium. In addition, the induction of phosphorylation of intracellular Smad-1/5 and Erk-1/2 proteins clearly supported the osteogenetic capacity of the biomaterial. Full article

The highly repetitive and variable fibroin heavy chain (FibH) gene can be used as a silkworm identification; however, only a few complete FibH sequences are known. In this study, we extracted and examined 264 FibH gene complete sequences (FibHome) from a high-resolution silkworm pan-genome. The average FibH lengths of the wild silkworm, local, and improved strains were 19,698 bp, 16,427 bp, and 15,795 bp, respectively. All FibH sequences had a conserved 5′ and 3′ terminal non-repetitive (5′ and 3′ TNR, 99.74% and 99.99% identity, respectively) sequence and a variable repetitive core (RC). The RCs differed greatly, but they all shared the same motif. During domestication or breeding, the FibH gene mutated with hexanucleotide (GGTGCT) as the core unit. Numerous variations existed that were not unique to wild and domesticated silkworms. However, the transcriptional factor binding sites, such as fibroin modulator-binding protein, were highly conserved and had 100% identity in the FibH gene’s intron and upstream sequences. The local and improved strains with the same FibH gene were divided into four families using this gene as a marker. Family I contained a maximum of 62 strains with the optional FibH (Opti-FibH, 15,960 bp) gene. This study provides new insights into FibH variations and silkworm breeding. Full article