UNASSIGNED: Synthetic fibers have many benefits in clinical practice; however, they cause microplastic pollution, and their unaffordable price increases treatment costs. Natural silk fibers require biocompatibility assessment. This study investigated the effects of natural and synthetic fiber-reinforced composites (FRCs) on the cytotoxicity of fibroblast cell lines.
UNASSIGNED: Three commercial synthetic fibers (polyethylene, quartz, and E-glass) and two silk fibers from Bombyx mori and Samia ricini cocoons were employed. These fibers were made into FRC samples (n=6) by impregnation in flowable composite using a brass mold (25×2×2 mm). NIH/3T3 mouse fibroblasts were cultured in Dulbecco\'s modified eagle medium, supplemented, and seeded in 2×104 cells/mL. They were stored at 37 °C under 5% CO2 for 24 hours. The FRC samples were made into powder, eluted in dimethylsulfoxide, continued with PBS, supplemented with Dulbecco\'s modified eagle medium (DMEM), and exposed to cells for 24 hours. Blank (medium only) and control (cells and medium) samples were included. Subsequently, MTT was added for 4 h and read by enzyme-linked immunosorbent assay (λ=570 nm). Cell viability (%) was calculated and analyzed using one-way ANOVA (α=0.05).
UNASSIGNED: All groups of FRCs showed>80% cell viability. One-way ANOVA showed no significant difference between FRC groups regarding the viability of fibroblast cell lines (P>0.05).
UNASSIGNED: Both natural silk and synthetic fibers exhibit low cytotoxicity to fibroblast cell lines. B. mori and S. ricini silk fibers showed the potential to be used as alternative synthetic fibers.

Bombyx mori is a lepidopteran holometabolous insect with distinct developmental stages: egg, larvae, pupae, and adult. The lepidopteran insect undergoes major modifications in the central nervous system (CNS) so as to adapt to the lifestyle of these distinct stages with specific habitats and functions from voraciously feeding larval stages to flying reproductive adults via dormant pupal stages. Such transitions are linked to transcriptional, epigenetic, and translational complexities. Therefore, studying rhythmic gene expression in CNS of various developmental stages and the effects of antagonists on developmental hormones requires a very stable reference gene (RG). To facilitate rhythmic gene expression studies using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in B. mori and the effect of developmental hormone juvenile hormone (JH) and 20-hydroxy ecdysone hormone (20 HE), antagonists Precocene 1 and testosterone, respectively, were used. Eight candidate RGs, namely, Translational initiation factor 3 subunit 4 (TI3S4), Translational initiation factor 3 subunit 5 (TI3S5), Ribosomal protein subunit 7 (RPs7), TATA-binding protein association factor (TAF13), Translational initiation factor 4 A (TI4A), Ribosomal protein (RPL32), Elongation factor 1 (EF1), and Arginine kinase (AK), were assessed in the CNS of B. mori. The postembryonic developmental (PED) stages used were the fifth late larval instar, early pupa, mid pupa, late pupa, and adult. The assessments were done at four different time points, Zeitgeber time (ZT) 0, 6, 12, and 18, to find stability towards 24-h rhythmic expression. RefFinder, geNorm, and Ct value analysis were performed. RefFinder and geNORM studies suggested stability order as TI3S4 > TI3S5 > RPs7, but Ct value evaluation showed stability order as TI3S5 > TI3S4 > RPs7. We therefore demonstrated that TI3S4, TI3S5, and RPs7 can be used as RG in various PED stages in CNS of B. mori (Strain: CB-hybrid, PM×CSR2) towards studies with effects of JH and 20 HE antagonists.

UNASSIGNED: Pebrine, caused by microsporidium Nosema bombycis, is a devastating disease that causes serious economic damages to the sericulture industry. Studies on development of therapeutic and diagnostic options for managing pebrine in silkworms are very limited. Methionine aminopeptidase type 2 (MetAP2) of microsporidia is an essential gene for their survival and has been exploited as the cellular target of drugs such as fumagillin and its analogues in several microsporidia spp., including Nosema of honeybees.
UNASSIGNED: In the present study, using molecular and bioinformatics tools, we performed in-depth characterization and phylogenetic analyses of MetAP2 of Nosema bombycis isolated from Guangdong province of China.
UNASSIGNED: The full length of MetAP2 gene sequence of Nosema bombycis (Guangdong isolate) was found to be 1278 base pairs (bp), including an open reading frame of 1,077 bp, encoding a total of 358 amino acids. The bioinformatics analyses predicted the presence of typical alpha-helix structural elements, and absence of transmembrane domains and signal peptides. Additionally, other characteristics of a stable protein were also predicted. The homology-based 3D models of MetAP2 of Nosema bombycis (Guangdong isolate) with high accuracy and reliability were developed. The MetAP2 protein was expressed and purified. The observed molecular weight of MetAP2 protein was found to be ~43-45 kDa. The phylogenetic analyses showed that MetAP2 gene and amino acids sequences of Nosema bombycis (Guangdong isolate) shared a close evolutionary relationship with Nosema spp. of wild silkworms, but it was divergent from microsporidian spp. of other insects, Aspergillus spp., Saccharomyces cerevisiae, and higher animals including humans. These analyses indicated that the conservation and evolutionary relationships of MetAP2 are closely linked to the species relationships.
UNASSIGNED: This study provides solid foundational information that could be helpful in optimization and development of diagnostic and treatment options for managing the threat of Nosema bombycis infection in sericulture industry of China.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Lead (Pb) is a major source of heavy metal contamination, and poses a threat to biodiversity and human health. Elevated levels of Pb can hinder insect growth and development, leading to apoptosis via mechanisms like oxidative damage. The midgut of silkworms is the main organ exposed to heavy metals. As an economically important lepidopteran model insect in China, heavy metal-induced stress on silkworms causes considerable losses in sericulture, thereby causing substantial economic damage. This study aimed to investigate Pb-induced detoxification-related genes in the midgut of silkworms using high-throughput sequencing methods to achieve a deeper comprehension of the genes\' reactions to lead exposure. This study identified 11,567 unigenes and 14,978 transcripts. A total of 1265 differentially expressed genes (DEGs) were screened, comprising 907 upregulated and 358 downregulated genes. Subsequently, Gene Ontology (GO) classification analysis revealed that the 1265 DEGs were distributed across biological processes, cellular components, and molecular functions. This suggests that the silkworm midgut may affect various organelle functions and biological processes, providing crucial clues for further exploration of DEG function. Additionally, the expression levels of 12 selected detoxification-related DEGs were validated using qRT-PCR, which confirmed the reliability of the RNA-seq results. This study not only provides new insights into the detoxification defense mechanisms of silkworms after Pb exposure, but also establishes a valuable foundation for further investigation into the molecular detoxification mechanisms in silkworms.

Mercury (Hg) contamination poses a global threat to the environment, given its elevated ecotoxicity. Herein, we employed the lepidopteran model insect, silkworm (Bombyx mori), to systematically investigate the toxic effects of Hg-stress across its growth and development, histomorphology, antioxidant enzyme activities, and transcriptome responses. High doses of Hg exposure induced evident poisoning symptoms, markedly impeding the growth of silkworm larvae and escalating mortality in a dose-dependent manner. Under Hg exposure, the histomorphology of both the midgut and fat body exhibited impairments. Carboxylesterase (CarE) activity was increased in both midgut and fat body tissues responding to Hg treatment. Conversely, glutathione S-transferase (GST) levels increased in the fat body but decreased in the midgut. The transcriptomic analysis revealed that the response induced by Hg stress involved multiple metabolism processes. Significantly differently expressed genes (DEGs) exhibited strong associations with oxidative phosphorylation, nutrient metabolisms, insect hormone biosynthesis, lysosome, ribosome biogenesis in eukaryotes, and ribosome pathways in the midgut or the fat body. The findings implied that exposure to Hg might induce the oxidative stress response, attempting to compensate for impaired metabolism. Concurrently, disruptions in nutrient metabolism and insect hormone activity might hinder growth and development, leading to immune dysfunction in silkworms. These insights significantly advance our theoretical understanding of the potential mechanisms underlying Hg toxicity in invertebrate organisms.

The domestic silkworm, Bombyx mori, has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass produce recombinant bioactive proteins containing silk-based materials. Furthermore, silkworms are well-known as a source of food and have also been orally administered to prevent and treat several human disorders. In this study, we aimed to investigate the inherent bio-physicochemical properties of edible silkworms to accurately evaluate their clinical and nutritional potential. We prepared raw powder from whole larvae of silkworm. The yield rate of the powder derived from dried larvae was almost 100% (98.1-99.1% in replicates). As \"percentage yield\" translates to \"Budomari\" in Japanese, this raw powder was named \"B100rw.\" We further prepared B100dn that was denatured through autoclaving. Thereafter, we examined whether B100rw sustained the original bio-physicochemical properties by comparing it with B100dn. There was no significant difference in nutritional content between B100rw and B100dn. B100rw contained proteins derived from silkworm larvae and mulberry leaves, whereas the proteins of B100dn were mostly degraded. On measuring the enzymatic activity of both powders using trehalase as an indicator enzyme, B100rw was found to maintain trehalase activity. B100rw also maintained a random coil conformation, similar to that of liquid silk. This suggested that B100rw sustained the unique bio-physicochemical properties of living larvae. These findings may facilitate the development of novel food products or orally administered vaccines.

UNASSIGNED: Pseudogenes are sequences that have lost the ability to transcribe RNA molecules or encode truncated but possibly functional proteins. While they were once considered to be meaningless remnants of evolution, recent researches have shown that pseudogenes play important roles in various biological processes. However, the studies of pseudogenes in the silkworm, an important model organism, are limited and have focused on single or only a few specific genes.
UNASSIGNED: To fill these gaps, we present a systematic genome-wide studies of pseudogenes in the silkworm.
UNASSIGNED: We identified the pseudogenes in the silkworm using the silkworm genome assemblies, transcriptome, protein sequences from silkworm and its related species. Then we used transcriptome datasets from 832 RNA-seq analyses to construct spatio-temporal expression profiles for these pseudogenes. Additionally, we identified tissue-specifically expressed and differentially expressed pseudogenes to further understand their characteristics. Finally, the functional roles of pseudogenes as lncRNAs were systematically analyzed.
UNASSIGNED: We identified a total of 4410 pseudogenes, which were grouped into 4 groups, including duplications (DUPs), unitary pseudogenes (Unitary), processed pseudogenes (retropseudogenes, RETs), and fragments (FRAGs). The most of pseudogenes in the domestic silkworm were generated before the divergence of wild and domestic silkworm, however, the domestication may also involve in the accumulation of pseudogenes. These pseudogenes were clearly divided into 2 cluster, a highly expressed and a lowly expressed, and the posterior silk gland was the tissue with the most tissue-specific pseudogenes (199), implying these pseudogenes may be involved in the development and function of silkgland. We identified 3299 lncRNAs in these pseudogenes, and the target genes of these lncRNAs in silkworm pseudogenes were enriched in the egg formation and olfactory function.
UNASSIGNED: This study replenishes the genome annotations for silkworm, provide valuable insights into the biological roles of pseudogenes. It will also contribute to our understanding of the complex gene regulatory networks in the silkworm and will potentially have implications for other organisms as well.

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL-CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL-CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

Genetic mutations leading to premature termination codons are known to have detrimental effects. Using the Lepidoptera model insect, the silkworm (Bombyx mori), we explored the genetic compensatory response triggered by mutations with premature termination codons. Additionally, we delved into the molecular mechanisms associated with the nonsense-mediated mRNA degradation pathway. CRISPR/Cas9 technology was utilized to generate a homozygous bivoltine silkworm line BmTrpA1-/- with a premature termination. Transcript levels were assessed for the BmTrpA paralogs, BmPyrexia and BmPainless as well as for the essential factors Upf1, Upf2, and Upf3a involved in the nonsense-mediated mRNA degradation (NMD) pathway. Upf2 was specifically knocked down via RNA interference at the embryonic stage. The results comfirmed that the BmTrpA1 transcripts with a 2-base deletion generating a premature termination codon in the BmTrpA1-/- line. From day 6 of embryonic development, the mRNA levels of BmPyrexia, BmPainless, Upf1, and Upf2 were significantly elevated in the gene-edited line. Embryonic knockdown of Upf2 resulted in the suppression of the genetic compensation response in the mutant. As a result, the offspring silkworm eggs were able to hatch normally after 10 days of incubation, displaying a non-diapause phenotype. It was observed that a genetic compensation response does exist in BmTrpA1-/-B. mori. This study presents a novel discovery of the NMD-mediated genetic compensation response in B. mori. The findings offer new insights into understanding the genetic compensation response and exploring the gene functions in lepidopteran insects, such as silkworms.