Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae.

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

The BTB-ZF gene Bm-mamo regulates pigmentation in silkworm caterpillars.

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor of dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermal features in some insects.

Photoperiodism of Diapause Induction in the Silkworm, Bombyx mori.

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.

BmSPP is a virus resistance gene in Bombyx mori.

Introduction: Signal peptide peptidase (SPP) is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although SPP is essential for many viral infections, its role in silkworms remains unclear. Studying its role in the silkworm, Bombyx mori , may be helpful in breeding virus-resistant silkworms. Methods: First, we performed RT-qPCR to analyze the expression pattern of BmSPP. Subsequently, we inhibited BmSPP using the SPP inhibitor 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leucylaminopropanone ((Z-LL)2-ketone) and downregulated the expression of BmSPP using CRISPR/Cas9 gene editing. Furthermore, we assessed the impact of these interventions on the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV). Results: We observed a decreased in the expression of BmSPP during viral proliferation. It was found that higher concentration of the inhibitor resulted in greater inhibition of BmNPV proliferation. The down-regulation of BmSPP in both in vivo and in vitro was found to affect the proliferation of BmNPV. In comparison to wild type silkworm, BmSPPKO silkworms exhibited a 12.4% reduction in mortality rate. Discussion: Collectively, this work demonstrates that BmSPP plays a negative regulatory role in silkworm resistance to BmNPV infection and is involved in virus proliferation and replication processes. This finding suggests that BmSPP servers as a target gene for BmNPV virus resistance in silkworms and can be utilized in resistance breeding programs.

Recent advances in the biosensors application for reviving infectious disease management in silkworm model: a new way to combat microbial pathogens.

Silkworms are an essential economic insect but are susceptible to diseases during rearing, leading to yearly losses in cocoon production. While chemical control is currently the primary method to reduce disease incidences, its frequent use can result in loss of susceptibility to pathogens and, ultimately, antibiotic resistance. To effectively prevent or control disease, growers must accurately, sensitively, and quickly detect causal pathogens to determine the best management strategies. Accurate recognition of diseased silkworms can prevent pathogen transmission and reduce cocoon loss. Different pathogen detection methods have been developed to achieve this objective, but they need more precision, specificity, consistency, and promptness and are generally unsuitable for in-situ analysis. Therefore, detecting silkworm diseases under rearing conditions is still an unsolved problem. As a consequence of this, there is an enormous interest in the development of biosensing systems for the early and precise identification of pathogens. There is also significant room for improvement in translating novel biosensor techniques to identify silkworm pathogens. This study explores the types of silkworm diseases, their symptoms, and their causal microorganisms. Moreover, we compare the traditional approaches used in silkworm disease diagnostics along with the latest sensing technologies, with a precise emphasis on lateral flow assay-based biosensors that can detect and manage silkworm pathogens.

Indoxacarb triggers autophagy and apoptosis through ROS accumulation mediated by oxidative phosphorylation in the midgut of Bombyx mori.

Indoxacarb has been widely utilized in agricultural pest management, posing a significant ecological threat to Bombyx mori, a non-target economic insect. In the present study, short-term exposure to low concentration of indoxacarb significantly suppressed the oxidative phosphorylation pathway, and resulted in an accumulation of reactive oxygen species (ROS) in the midgut of B. mori. While, the ATP content exhibited a declining trend but there was no significant change. Moreover, indoxacarb also significantly altered the transcription levels of six autophagy-related genes, and the transcription levels of ATG2, ATG8 and ATG9 were significantly up-regulated by 2.56-, 1.90-, and 3.36-fold, respectively. The protein levels of ATG8-I and ATG8-II and MDC-stained frozen sections further suggested an increase in autophagy. Furthermore, the protein level and enzyme activity of CASP4 showed a significant increase in accordance with the transcription levels of apoptosis-related genes, indicating the activation of the apoptotic signaling pathway. Meanwhile, the induction of apoptosis signals in the midgut cells triggered by indoxacarb was confirmed through TUNEL staining. These findings suggest that indoxacarb can promote the accumulation of ROS by inhibiting the oxidative phosphorylation pathway, thereby inducing autophagy and apoptosis in the midgut cells of B. mori.

The Potential of Natural Carotenoids-Containing Sericin of the Domestic Silkworm Bombyx mori.

Sericin derived from the white cocoon of Bombyx mori has been attracting more attention for its utilization in food, cosmetics, and biomedicine. The potential health benefits of natural carotenoids for humans have also been well-established. Some rare strains of Bombyx mori (B. mori) produce yellow-red cocoons, which endow a potential of natural carotenoid-containing sericin. We hypothesized that natural carotenoid-containing sericin from yellow-red cocoons would exhibit better properties compared with white cocoon sericin. To investigate the physicochemical attributes of natural carotenoid-containing sericin, we bred two silkworm strains from one common ancestor, namely XS7 and XS8, which exhibited different cocoon colors as a result of the inconsistent distribution of lutein and ß-carotene. Compared with white cocoon sericin, the interaction between carotenoids and sericin molecules in carotenoid-containing sericin resulted in a unique fluorescence emission at 530, 564 nm. The incorporation of carotenoids enhanced the antibacterial effect, anti-cancer ability, cytocompatibility, and antioxidant of sericin, suggesting potential wide-ranging applications of natural carotenoid-containing sericin as a biomass material. We also found differences in fluorescence characteristics, antimicrobial effects, anti-cancer ability, and antioxidants between XS7 and XS8 sericin. Our work for the first time suggested a better application potential of natural carotenoid-containing sericin as a biomass material than frequently used white cocoon sericin.

Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Safe utilization and remediation potential of the mulberry-silkworm system in heavy metal-contaminated lands: A review.

Mulberry cultivation and silkworm rearing hold a prominent position in the agricultural industries of many Asian countries, contributing to economic growth, sustainable development, and cultural heritage preservation. Applying the soil-mulberry-silkworm system (SMSS) to heavy metal (HM)-contaminated areas is significant economically, environmentally, and socially. The ultimate goal of this paper is to review the main research progress of SMSS under HM stress, examining factors affecting its safe utilization and remediation potential for HM-contaminated soils. HM tolerance of mulberry and silkworms relates to their growth stages. Based on the standards for HM contaminants in various mulberry and silkworm products and the bioconcentration factor of HMs at different parts of SMSS, we calculated maximum safe Cd and Pb levels for SMSS application on contaminated lands. Several remediation practices demonstrated mulberry's ability to grow on barren lands, absorb various HMs, while silkworm excreta can adsorb HMs and improve soil fertility. Considering multiple factors influencing HM tolerance and accumulation, we propose a decision model to guide SMSS application in polluted areas. Finally, we discussed the potential of using molecular breeding techniques to screen or develop varieties better suited for HM-contaminated regions. However, actual pollution scenarios are often complex, requiring consideration of multiple factors. More large-scale applications are crucial to enhance the theoretical foundation for applying SMSS in HM pollution risk areas.

[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Domestication Gene Mlx and Its Partner Mondo Are Involved in Controlling the Larval Body Size and Cocoon Shell Weight of Bombyx mori.

Bombyx mori was domesticated from Bombyx mandarina. The long-term domestication of the silkworm has brought about many remarkable changes to its body size and cocoon shell weight. However, the molecular mechanism underlying the improvement in the economic characteristics of this species during domestication remains unclear. In this study, we found that a transposable element (TE)-Bm1-was present in the upstream regulatory region of the Mlx (Max-like protein X) gene in wild silkworms but not in all domesticated silkworms. The absence of Bm1 caused an increase in the promoter activity and mRNA content of Mlx. Mlx and its partner Mondo belong to the bHLHZ transcription factors family and regulate nutrient metabolism. RNAi of Mlx and Mondo decreased the expression and promoter activity of glucose metabolism-related genes (trehalose transport (Tret), phosphofructokinase (PFK), and pyruvate kinase (PK)), lipogenic genes (Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)), and glutamine synthesis gene (Glutamine synthase 2, (GS2)). Furthermore, the transgenic overexpression of Mlx and Mondo in the fat body of silkworms increased the larval body size, cocoon shell weight, and egg number, but the silencing of the two genes resulted in the opposite phenotypes. Our results reveal the molecular mechanism of Mlx selection during domestication and its successful use in the molecular breeding of Bombyx mori.

Molecular mechanism analysis of apoptosis induced by silk fibroin peptides.

Silk fibroin derived from silkworm cocoons exhibits excellent mechanical properties, good biocompatibility, and low immunogenicity. Previous studies showed that silk fibroin had an inhibitory effect on cells, suppressing proliferation and inducing apoptosis. However, the source of the toxicity and the mechanism of apoptosis induction are still unclear. In this study, we hypothesized that the toxicity of silk fibroin might originate from the crystalline region of the heavy chain of silk fibroin. We then verified the hypothesis and the specific induction mechanism. A target peptide segment was obtained from α-chymotrypsin. The potentially toxic mixture of silk fibroin peptides (SFPs) was separated by ion exchange, and the toxicity was tested by an MTT assay. The results showed that SFPs obtained after 4 h of enzymatic hydrolysis had significant cytotoxicity, and SFPs with isoelectric points of 4.0-6.8 (SFPα II) had a significant inhibitory effect on cell growth. LC-MS/MS analysis showed that SFPα II contained a large number of glycine-rich and alanine-rich repetitive sequence polypeptides from the heavy-chain crystallization region. A series of experiments showed that SFPα II mediated cell death through the apoptotic pathway by decreasing the expression of Bcl-2 protein and increasing the expression of Bax protein. SFPα II mainly affected the p53 pathway and the AMPK signaling pathway in HepG2 cells. SFPα II may indirectly increase the expression of Cers2 by inhibiting the phosphorylation of EGFR, which activated apoptotic signaling in the cellular mitochondrial pathway and inhibited the Akt/NF-κB pathway by increasing the expression of PPP2R2A.

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Thermochemical conversion of silkworm by-product into syngas.

This study explored the valorisation of silkworm by-product, a major by-product of the silk industry (sericulture), which amounts to 16 million tonnes annually. The focus was on transforming waste into energy resources through pyrolysis under CO2 conditions. In one-stage pyrolysis, the evolution of syngas under N2 was found to be comparable to that under CO2. A notable allocation of carbon to biocrude rather than syngas was observed. The two-stage pyrolysis resulted in increased syngas production. However, achieving a homogeneous reaction between CO2 and the volatiles liberated from silkworm byproduct proved challenging. Indeed, the reaction kinetics governing CO2 reactivity was not fast although the temperature windows of the reaction were aligned in the two-stage pyrolysis. To address this issue, pyrolysis was performed using a Ni-based catalyst to expedite the reaction kinetics. Consequently, syngas formation, particularly CO formation, was significantly enhanced under CO2 conditions compared to that under N2 conditions. The syngas yield under CO2 was 36.42 wt% which was 2-fold higher than that of N2. This suggested the potential of CO2 altering the carbon distribution from biocrude to syngas. This strategy would contribute to the establishment of sustainable production of silk by converting sericulture by-product into energy/chemical resources.

Injectable Bombyx mori (B. mori) silk fibroin/MXene conductive hydrogel for electrically stimulating neural stem cells into neurons for treating brain damage.

Brain damage is a common tissue damage caused by trauma or diseases, which can be life-threatening. Stem cell implantation is an emerging strategy treating brain damage. The stem cell is commonly embedded in a matrix material for implantation, which protects stem cell and induces cell differentiation. Cell differentiation induction by this material is decisive in the effectiveness of this treatment strategy. In this work, we present an injectable fibroin/MXene conductive hydrogel as stem cell carrier, which further enables in-vivo electrical stimulation upon stem cells implanted into damaged brain tissue. Cell differentiation characterization of stem cell showed high effectiveness of electrical stimulation in this system, which is comparable to pure conductive membrane. Axon growth density of the newly differentiated neurons increased by 290% and axon length by 320%. In addition, unfavored astrocyte differentiation is minimized. The therapeutic effect of this system is proved through traumatic brain injury model on rats. Combined with in vivo electrical stimulation, cavities formation is reduced after traumatic brain injury, and rat motor function recovery is significantly promoted.

Impact of the Reduction Time-Dependent Electrical Conductivity of Graphene Nanoplatelet-Coated Aligned Bombyx mori Silk Scaffolds on Electrically Stimulated Axonal Growth.

Graphene-based nanomaterials, renowned for their outstanding electrical conductivity, have been extensively studied as electroconductive biomaterials (ECBs) for electrically stimulated tissue regeneration. However, using eco-friendly reducing agents like l-ascorbic acid (l-Aa) can result in lower conductive properties in these ECBs, limiting their full potential for smooth charge transfer in living tissues. Moreover, creating a flexible biomaterial scaffold using these materials that accurately mimics a specific tissue microarchitecture, such as nerves, poses additional challenges. To address these issues, this study developed a microfibrous scaffold of Bombyx mori (Bm) silk fibroin uniformly coated with graphene nanoplatelets (GNPs) through a vacuum coating method. The scaffold's electrical conductivity was optimized by varying the reduction period using l-Aa. The research systematically investigated how different reduction periods impact scaffold properties, focusing on electrical conductivity and its significance on electrically stimulated axonal growth in PC12 cells. Results showed that a 48 h reduction significantly increased surface electrical conductivity by 100-1000 times compared to a shorter or no reduction process. l-Aa contributed to stabilizing the reduced GNPs, demonstrated by a slow degradation profile and sustained conductivity even after 60 days in a proteolytic environment. ß (III) tubulin immunostaining of PC12 cells on varied silk:GNP scaffolds under pulsed electrical stimulation (ES, 50 Hz frequency, 1 ms pulse width, and amplitudes of 100 and 300 mV/cm) demonstrates accelerated axonal growth on scaffolds exhibiting higher conductivity. This is supported by upregulated intracellular Ca2+ dynamics immediately after ES on the scaffolds with higher conductivity, subjected to a prolonged reduction period. The study showcases a sustainable reduction approach using l-Aa in combination with natural Bm silk fibroin to create a highly conductive, mechanically robust, and stable silk:GNP-based aligned fibrous scaffold. These scaffolds hold promise for functional regeneration in electrically excitable tissues such as nerves, cardiac tissue, and muscles.

Biological activity of silver nanoparticles synthesized using viticultural waste.

This research paper presents a novel approach to the green synthesis of silver nanoparticles (AgNPs) using viticultural waste, allowing to obtain NP dispersions with distinct properties and morphologies (monodisperse and polydisperse AgNPs, referred to as mAgNPs and pAgNPs) and to compare their biological activities. Our synthesis method utilized the ethanolic extract of Vitis vinifera pruning residues, resulting in the production of mAgNPs and pAgNPs with average sizes of 12 ± 5 nm and 19 ± 14 nm, respectively. Both these AgNPs preparations demonstrated an exceptional stability in terms of size distribution, which was maintained for one year. Antimicrobial testing revealed that both types of AgNPs inhibited either the growth of planktonic cells or the metabolic activity of biofilm sessile cells in Gram-negative bacteria and yeasts. No comparable activity was found towards Gram-positives. Overall, pAgNPs exhibited a higher antimicrobial efficacy compared to their monodisperse counterparts, suggesting that their size and shape may provide a broader spectrum of interactions with target cells. Both AgNP preparations showed no cytotoxicity towards a human keratinocyte cell line. Furthermore, in vivo tests using a silkworm animal model indicated the biocompatibility of the phytosynthesized AgNPs, as they had no adverse effects on insect larvae viability. These findings emphasize the potential of targeted AgNPs synthesized from viticultural waste as environmentally friendly antimicrobial agents with minimal impact on higher organisms.

Design of Bombyx mori (B. mori) Silk Fibroin Microspheres for Developing Biosafe Sunscreen.

Sunscreens play a crucial role in protecting the skin from ultraviolet (UV) damage. However, present commercial sunscreens have a tendency to generate free radicals in the UV window, resulting in serious inflammatory responses and health problems. In this study, we demonstrate that silk fibroin microspheres (SFMPs) assembled from regenerated silk fibroin (SF) could scavenge free radicals while preventing UV irradiation and thus present a promising sunscreen. The SFMP reflected more UV light than SF and presented a higher stability than that of organic commercial sunscreens. In vitro analysis proved that SFMP could more efficiently scavenge the hydroxy radical and reduce the intracellular reactive oxygen than titanium dioxide (TiO2). In vivo experiments exhibited that SFMP provided stronger skin protection against UV irradiation than commercial sunscreens and TiO2. Furthermore, SFMP treatment significantly inhibited the skin inflammatory response. This work suggests that the SFMP has great potential to be developed into a biosafe sunscreen.

Harvesting Silk Fibers from Plodia interpunctella: Role of Environmental Rearing Conditions in Fiber Production and Properties.

Silk fibers are produced by a wide variety of insects. The silkworm Bombyx mori (Bombyx) was domesticated because the physical properties of its silk fibers were amenable to the production of fine textiles. Subsequently, engineers have regenerated silk fibroin to form biomaterials. The monocular focus on Bombyx silk has underutilized the expanse of diverse silk proteins produced by more than 100,000 other arthropods. This vast array of silk fibers could be utilized for biomedical engineering challenges if sufficient rearing and purification processes are developed. Herein, we show that the moth, Plodia interpunctella (Plodia), represents an alternative silk source that is easily reared in highly regulated culture environments allowing for greater consistency in the silk produced. We controlled the temperature, resource availability (larvae/gram diet), and population density (larvae/mL) with the goal of increasing silk fiber production and improving homogeneity in Plodia silk proteins. We determined that higher temperatures accelerated insect growth and reduced life cycle length. Furthermore, we established initial protocols for the production of Plodia silk with optimal silk production occurring at 24 °C, with a resource availability of 10 larvae/gram and a population density of 0.72 larvae/mL. Population density was shown to be the most prominent driving force of Plodia silk mat formation among the three parameters assessed. Future work will need to link gene expression, protein production and purification, and resulting mechanical properties as a function of environmental cues to further transition Plodia silk into regenerated silk fibroin biomaterials.