Silk Sericin and Its Composite Materials with Antibacterial Properties to Enhance Wound Healing: A Review.

Wound infections may disrupt the normal wound-healing process. Large amounts of antibiotics are frequently used to prevent pathogenic infections; however, this can lead to resistance development. Biomaterials possessing antimicrobial properties have promising applications for reducing antibiotic usage and promoting wound healing. Silk sericin (SS) has been increasingly explored for skin wound healing applications owing to its excellent biocompatibility and antioxidant, antimicrobial, and ultraviolet-resistant properties. In recent years, SS-based composite biomaterials with a broader antimicrobial spectrum have been extensively investigated and demonstrated favorable efficacy in promoting wound healing. This review summarizes various antimicrobial agents, including metal nanoparticles, natural extracts, and antibiotics, that have been incorporated into SS composites for wound healing and elucidates their mechanisms of action. It has been revealed that SS-based biomaterials can achieve sustained antimicrobial activity by slow-release-loaded antimicrobial agents. The antimicrobial-loaded SS composites may promote wound healing through anti-infection, anti-inflammation, hemostasis, angiogenesis, and collagen deposition. The manufacturing methods, benefits, and limitations of antimicrobial-loaded SS materials are briefly discussed. This review aims to enhance the understanding of new advances and directions in SS-based antimicrobial composites and guide future biomedical research.

Advances of regenerated and functionalized silk biomaterials and application in skin wound healing.

The cocoon silk of silkworms (Bombyx mori) has multiple potential applications in biomedicine due to its good biocompatibility, mechanical properties, degradability, and plasticity. Numerous studies have confirmed that silk material dressings are more effective than traditional ones in the skin wound healing process. Silk material research has recently moved toward functionalized biomaterials and achieved remarkable results. Herein, we summarize the recent advances in functionalized silk materials and their efficacy in skin wound healing. In particular, transgenic technology has realized the specific expression of human growth factors in the silk glands of the silkworms, which lays the foundation for fabricating novel and low-cost functionalized materials. Without a green and safe preparation process, the best raw silk materials cannot be made into medically safe products. Therefore, we provide an overview of green and gentle approaches for silk degumming and silk sericin (SS) extraction. Moreover, we summarize and discuss the processing methods of silk fibroin (SF) and SS materials and their potential applications, such as burns, diabetic wounds, and other wounds. This review aims to enhance our understanding of new advances and directions in silk materials and guide future biomedical research.

Effects of short-term exposure to volatile pesticide dichlorvos on the olfactory systems in Spodoptera litura: Calcium homeostasis, synaptic plasticity and apoptosis.

Volatile pesticides are a growing environmental and public health concern. However, little attention has been paid to its olfactory neurotoxic effect on pests and non-target organisms. Dichlorvos is a widely used organophosphorus fumigant that is ubiquitous in the environment. This study aims to explore the mode of action of the volatile dichlorvos-mediated olfactory impairment using a lepidopteran insect Spodoptera litura as a model. It was indicated that electroantennogram amplitudes of the male moths' response to sex pheromones and phenylacetaldehyde were reduced by approximately 20 % after 12-h fumigation exposure. RNA-Sequencing analysis revealed that down-regulation of trypsin and CLIC2 might be responsible for inhibition of odor recognition in the antenna, the peripheral olfactory tissue. In the head, 822 (84.05 %) of the 978 differentially expressed genes (DEGs) were up-regulated, of which seven DEGs encoding transcription factors may mainly modulate the stress-regulatory networks. Combining transcriptome with brain calcium imaging and Annexin V-mCherry staining experiments showed that volatile dichlorvos mainly disrupts Ca2+ homeostasis and synaptic plasticity, induces apoptosis in the central nervous system, and further leads to olfactory dysfunction. Overall, this study highlighted a comprehensive work model for dichlorvos-induced olfactory impairment in S. litura and may provide insights into toxic effects of airborne organophosphates on non-target organisms.

Olfactory dysfunction and potential mechanisms caused by volatile organophosphate dichlorvos in the silkworm as a model animal.

Volatile pesticides impair olfactory function in workers/farmers and insects, but data on molecular responses and mechanisms are poorly understood. This study aims to reveal the mechanisms of olfactory dysfunction in the silkworm after exposure to volatile dichlorvos. Our results demonstrated that acute exposure for 12 h significantly reduced electroantennogram responses, and over 62.50% of the treated male moths cannot locate the pheromone source. Transcriptional and proteomic responses of the antennae and heads were investigated. A total of 101 differentially expressed genes (DEGs) in the antennae, 138 DEGs in the heads, and 43 differentially expressed proteins (DEPs) in the heads including antennae were revealed. We discovered that upregulations of Arrestin1 and nitric oxide synthase1 (NOS1) may inhibit cyclic nucleotide-gated channels and hinder calcium influx in the antennae. In the central nervous systems (CNS), downregulations of tyrosine hydroxylase (TH) and tyrosine decarboxylase (TDC) may inhibit olfactory signal transduction by reducing the second messenger biosynthesis. Meanwhile, an abnormal increase of brain cell apoptosis was revealed by Annexin V-mCherry staining, often leading to persistent neurologic impairment. Taken together, this study highlighted olfactory dysfunction caused by dichlorvos, which may provide a novel perspective for understanding the toxicity mechanism of volatile pesticides in other organisms.

Heat Shock Protein 70 Family in Response to Multiple Abiotic Stresses in the Silkworm.

The 70 kDa heat shock proteins play important roles in protecting organisms against environmental stresses, which are divided into stress-inducible forms (HSP70s) and heat shock cognates (HSC70s). In this study, heat shock protein 70 family was identified in the whole genome of the silkworm. Based on the known nomenclature and phylogenetic analysis, four HSP70s and five HSC70s were classified. Relatively, heat shock cognates were more conservative and were constitutively expressed in various tissues of the silkworm larvae. Under thermal (37 °C and 42 °C) and cold (2 °C) stresses, the expressions of HSP70-1, HSP70-2, and HSP70-3 were up-regulated, and the highest induction reached 4147.3, 607.1, and 1987.3 times, respectively. Interestingly, HSC70-1, HSC70-4, and HSC70-5 also showed slight induced expressions in the fat body and/or midgut under thermal stresses. In addition, the expression of HSP70-1 was induced by dichlorvos and phoxim insecticides, while most HSC70 genes were inhibited. The results suggested that stress-inducible forms play more important roles in adaptation to various stresses than HSC70s.

Genome-wide identification and analysis of the thiolase family in insects.

Thiolases are important enzymes involved in lipid metabolism in both prokaryotes and eukaryotes, and are essential for a range of metabolic pathways, while, little is known for this important family in insects. To shed light on the evolutionary models and functional diversities of the thiolase family, 137 thiolase genes were identified in 20 representative insect genomes. They were mainly classified into five classes, namely cytosolic thiolase (CT-thiolase), T1-thiolase, T2-thiolase, trifunctional enzyme thiolase (TFE-thiolase), and sterol carrier protein 2 thiolase (SCP2-thiolase). The intron number and exon/intron structures of the thiolase genes reserve large diversification. Subcellular localization prediction indicated that all the thiolase proteins were mitochondrial, cytosolic, or peroxisomal enzymes. Four highly conserved sequence fingerprints were found in the insect thiolase proteins, including CxS-, NEAF-, GHP-, and CxGGGxG-motifs. Homology modeling indicated that insect thiolases share similar 3D structures with mammals, fishes, and microorganisms. In Bombyx mori, microarray data and reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that some thiolases might be involved in steroid metabolism, juvenile hormone (JH), and sex pheromone biosynthesis pathways. In general, sequence and structural characteristics were relatively conserved among insects, bacteria and vertebrates, while different classes of thiolases might have differentiation in specific functions and physiological processes. These results will provide an important foundation for future functional validation of insect thiolases.

Identification of genes involved in sex pheromone biosynthesis and metabolic pathway in the Chinese oak silkworm, Antheraea pernyi.

The Chinese oak silkworm, Antheraea pernyi, has not only been semi-domesticated as an important economical insect but also used for genetic research. The female moths of A. pernyi employ a pheromone blend containing (E,Z)-6,11-hexadecadienal (E6,Z11-16:Ald), (E,Z)-6,11-hexadecadienyl acetate (E6,Z11-16:OAc), and (E,Z)-4,9-tetradecadienyl acetate (E4,Z9-14:OAc). While its biosynthesis pathway is largely unknown. By deep sequencing and de novo assembly of sex pheromone gland (PG) transcriptome, we identified 141 candidate genes that are putatively related to pheromone biosynthesis, degradation, and chemoreception in A. pernyi. Gene expression patterns and phylogenetic analysis revealed that two desaturases (AperDES1 and 2), two fatty acid reductase (AperFAR1 and 2), and three acetyltransferase genes (AperACT1, 2 and 3) showed PG-biased or specific expression and were phylogenetically related to genes known to be involved in pheromone synthesis in other species. Furthermore, two carboxylesterases (AperCOE6 and 11) and two chemosensory protein (AperCSP1 and 6) were also expressed specifically or predominantly in the PGs, which might be related to sex pheromone degradation and transportation, respectively. Based on these results, the sex pheromone biosynthesis and metabolic pathway was proposed in A. pernyi. This study provides some crucial candidates for further functional elucidation, and may be used for interfering sexual communication in other Saturniidae pests.

Genetic and genomic analysis for cocoon yield traits in silkworm.

Domestic species provides a powerful model for examining genetic mechanisms in the evolution of yield traits. The domestic silkworm (Bombyx mori) is an important livestock species in sericulture. While the mechanisms controlling cocoon yield are largely unknown. Here, using B. mori and its wild relative B. mandarina as intercross parents, 100 BC1 individuals were sequenced by restriction site-associated DNA sequencing (RAD-Seq). The linkage map contained 9,632 markers was constructed. We performed high-resolution quantitative trait locus (QTL) mapping for four cocoon yield traits. A total of 11 QTLs were identified, including one yield-enhancing QTL from wild silkworm. By integrating population genomics and transcriptomic analysis with QTLs, some favourable genes were revealed, including 14 domestication-related genes and 71 differentially expressed genes (DEGs) in the fifth-instar larval silk gland transcriptome between B. mori and B. mandarina. The relationships between the expression of two important candidate genes (KWMTBOMO04917 and KWMTBOMO12906) and cocoon yield were supported by quantitative real-time PCR (qPCR). Our results provide some new insights into the molecular mechanisms of complex yield traits in silkworm. The combined method might be an efficient approach for identifying putative causal genes in domestic livestock and wild relatives.

Evidence of peripheral olfactory impairment in the domestic silkworms: insight from the comparative transcriptome and population genetics.

BACKGROUND: The insect olfactory system is a highly specific and sensitive chemical detector, which plays important roles in feeding, mating and finding an appropriate oviposition site. The ecological niche of Bombyx mori has changed greatly since domestication from B. mandarina, and its olfactory response to environmental odorants clearly decreased. However, the mechanisms that result in the olfactory impairment are largely unknown. RESULTS: The antennal transcriptomes were compared between the domestic and wild silkworms. Comparison of the same sex between the domestic and wild silkworms revealed 1410 and 1173 differentially expressed genes (DEGs) in males and females, respectively. To understand the olfactory impairment, we mainly focused on the olfactory-related genes. In total, 30 olfactory genes and 19 odorant-degrading enzymes (ODEs) showed differential expression in the two comparisons, in which 19 and 14 were down-regulated in the domestic silkworm, respectively. Based on population genomic data, the down-regulated odorant receptors (ORs) showed a higher ratio of unique non-synonymous polymorphisms to synonymous polymorphisms (N/S ratio) in the domestic populations than that in the wild silkworms. Furthermore, one deleterious mutation was found in OR30 of the domestic population, which was located in transmembrane helix 6 (TM6). CONCLUSIONS: Our results suggested that down-regulation of the olfactory-related genes and relaxed selection might be the major reasons for olfactory impairment of the domestic silkworm reared completely indoor environment. Reversely, wild silkworm may increase expression and remove deleterious polymorphisms of olfactory-related genes to retain sensitive olfaction.

Identification and comparison of long non-coding RNAs in the silk gland between domestic and wild silkworms.

Under long-term artificial selection, the domestic silkworm (Bombyx mori) has increased its silk yield tremendously in comparison with its wild progenitor, Bombyx mandarina. However, the molecular mechanism of silk yield increase is still unknown. Comparative analysis of long non-coding RNAs (lncRNAs) may provide some insights into understanding this phenotypic variation. In this study, using RNA sequencing technology data of silk gland in domestic and wild silkworms, we identified 599 lncRNAs in the silk gland of the silkworm. Compared with protein-coding genes, the silk gland lncRNA genes tend to have fewer exon numbers, shorter transcript length and lower GC-content. Moreover, we found that three lncRNA genes are significantly and differentially expressed between domestic and wild silkworms. The potential targets of two differentially expressed lncRNAs (DELs) (dw4sg_0040 and dw4sg_0483) and the expression-correlated genes with the two DELs are mainly enriched in the related processes of silk protein translation. This implies that these DELs may affect the phenotypic variation in silk yield between the domestic and wild silkworms through the post-transcriptional regulation of silk protein.

The transcriptome response of Heliconius melpomene larvae to a novel host plant.

In the warfare between herbivore and host plant, insects have evolved a variety of defensive mechanisms, including allelochemical transformation and excretion. Several studies have explored the transcriptome responses of insects after host plant shifts to understand these mechanisms. We investigated the plastic responses of Heliconius melpomene larvae feeding on a native host Passiflora menispermifolia and a less strongly defended nonhost species, Passiflora biflora. In total, 326 differentially expressed genes were identified, with a greater number upregulated on the more strongly defended native host. Functional annotation showed that detoxifying enzymes, transporters and components of peritrophic membrane were strongly represented. In total, 30 candidate detoxification genes were differentially expressed, with glutathione S-transferases (GSTs) and UDP-glucuronosyltransferases (UGTs) showing the highest proportion of differential expression, 27.3% and 17.3%, respectively. These differentially expressed detoxification genes were shown to evolve mainly under the influence of purifying selection, suggesting that protein-coding evolution has not played a major role in host adaptation. We found only one gene, GSTe3, with evidence of adaptive evolution at H40, which is around the G-site and might alter enzyme activity. Based on our transcriptome and molecular evolution analysis, we suggest that transcriptional plasticity of genes in a herbivore may play an important role in adaptation to a new host plant.

Characterization of an epsilon-class glutathione S-transferase involved in tolerance in the silkworm larvae after long term exposure to insecticides.

This study assessed the effect of the pesticides on activity and expression of glutathione S-transferases (GSTs) in the midgut of the silkworm after intense selections by phoxim and fenpropathrin for five and four generations, respectively. GSTs activity towards cumene hydroperoxide (CHP), a substrate of GSH-dependent peroxidase, was significantly increased in the crude homogenate of midguts after long term exposure to the insecticides. An epsilon-class GST gene (BmGSTe2) was identified and showed elevated expression in the midguts of phoxim- and fenpropathrin-selected strains. Expression of BmGSTe2 was only detected in the midgut at transcriptional and translational levels. The recombinant BmGSTE2 possessed peroxidase activity and significantly inhibited by fenpropathrin, phoxim and chlorpyrifos in vitro. These results indicated that BmGSTE2 might be one of the enzymes involved in enhancing larval tolerance to the insecticides used. Furthermore, GST activity and expression level of BmGSTe2 might be used as biomarkers of organophosphorus and pyrethroid insecticide exposures.

Comparative analysis of the silk gland transcriptomes between the domestic and wild silkworms.

BACKGROUND: Bombyx mori was domesticated from the Chinese wild silkworm, Bombyx mandarina. Wild and domestic silkworms are good models in which to investigate genes related to silk protein synthesis that may be differentially expressed in silk glands, because their silk productions are very different. Here we used the mRNA deep sequencing (RNA-seq) approach to identify the differentially expressed genes (DEGs) in the transcriptomes of the median/posterior silk glands of two domestic and two wild silkworms. RESULTS: The results indicated that about 58% of the total genes were expressed (reads per kilo bases per million reads (RPKM) ≥ 1) in each silkworm. Comparisons of the domestic and wild silkworm transcriptomes revealed 32 DEGs, of which 16 were up-regulated in the domestic silkworms compared with in the wild silkworms, and the other 16 were up-regulated in the wild silkworms compared with in the domestic silkworms. Quantitative real-time polymerase chain reaction (qPCR) was performed for 15 randomly selected DEGs in domestic versus wild silkworms. The qPCR results were mostly consistent with the expression levels determined from the RNA-seq data. Based on a Gene Ontology (GO) enrichment analysis and manual annotation, five of the up-regulated DEGs in the wild silkworms were predicted to be involved in immune response, and seven of the up-regulated DEGs were related to the GO term "oxidoreductase activity", which is associated with antioxidant systems. In the domestic silkworms, the up-regulated DEGs were related mainly to tissue development, secretion of proteins and metabolism. CONCLUSIONS: The up-regulated DEGs in the two domestic silkworms may be involved mainly in the highly efficient biosynthesis and secretion of silk proteins, while the up-regulated DEGs in the two wild silkworms may play more important roles in tolerance to pathogens and environment adaptation. Our results provide a foundation for understanding the molecular mechanisms of the silk production difference between domestic and wild silkworms.

Effect of organophosphate phoxim exposure on certain oxidative stress biomarkers in the silkworm.

Organophosphorus (OP) insecticides are widely used in agriculture, which are toxic to insect pests and nontarget organisms. The current study mainly assessed the effect of the pesticide phoxim on oxidative stress by certain biomarkers in the fat body and midgut of the silkworm, Bombyx mori (L.), after exposure to 50% lethal concentration (LC50) of phoxim for 2 h. Malondialdehyde (MDA) content, activity of glutathione transferase (GST), and expression of GST at transcriptional level were assayed. LC50 value of phoxim was 2.5 mg/liter at 2-h exposure for the day 3 of the fifth-instar larvae. After exposure of phoxim, MDA content in the fat body significantly increased at 4-20 h posttreatment (p.t.),the highest increase was approximately 4.11-fold from 0.451 +/- 0.053 to 1.854 +/- 0.113 nmol/mg protein compared with corresponding control. In the midgut, significant increase in the MDA content (from 1.40- to 3.16-fold) was observed at 8-42 h p.t. The activity of GSTs increased to 1.48-2.00-fold at 24-42 h p.t. and 1.33-1.48-fold at 20-24 h p.t. in the fat body and midgut, respectively. The peroxidase activity of GSTs also was induced, which increased to 1.46-2.06-fold and 1.31-1.50-fold in the fat body and midgut, respectively. BmGSTe8 showed a late up-regulation of transcripts at 24-42 h after exposure to phoxim, which might contribute to the improved phoxim tolerance of silkworm larvae. These results indicated that phoxim could trigger oxidative stress and that MDA content and GST activity might be used as biomarkers of OP insecticide exposure. In addition, activity of GSTs were more inducible in the fat body than in midgut.