The Bombyx mori singed Gene Is Involved in the High-Temperature Resistance of Silkworms.

Temperature is an important factor in the growth, development, survival, and reproduction of organisms. The high-temperature resistance mechanism of insects may be significant for use in the prevention and control of insect pests. The silkworm, Bombyx mori, is an important Lepidoptera model species for studies on pest control in agriculture and forestry. We identified a gene in B. mori, the B. mori singed (Bmsn) gene, which is involved in the high-temperature resistance of silkworms. Sn proteins are highly conserved among species in many taxonomic groups. The overexpression of the Bmsn gene promoted the proliferation of silkworm cells, reduced oxidation, and reduced the accumulation of reactive oxygen species under stress. Interfering with the Bmsn gene had the opposite result. We constructed a transgenic B. mori strain that overexpressed the Bmsn gene. The physiological traits of the transgenic strain were significantly improved, and it had stronger high-temperature resistance. The Bmsn gene is involved in the process by which fat bodies respond to high-temperature stress. These findings provide insights into the mechanism of high-temperature resistance of insects and offer a new perspective on agricultural and forestry pest control.

Microsporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission.

Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission.

Effects of artificial diet rearing during all instars on silk secretion and gene transcription in Bombyx mori (Lepidoptera: Bombycidae).

Silkworms (Bombyx mori) reared on artificial diets during all instars have the advantages of simplicity and efficiency, year-round production, and reduced risk of poisoning. However, low silk yield remains a challenge, limiting its industrial application. To address this issue, the spinning behavior, nutrient absorption, and transcriptomics of silkworms were investigated. Compared with silkworms reared on mulberry leaves during all instars, those fed with artificial diets showed significantly lower cocoon weight, cocoon shell weight, cocoon shell rate, and silk gland tissue somatic index at the end of the fifth instar (P < 0.01). The spinning duration and crawling distance of silkworms reared on artificial diets were also significantly lower than those reared on mulberry leaves (P < 0.01). Regarding nutrient absorption, the dietary efficiency indexes of silkworms fed with artificial diets were significantly lower than those fed with mulberry leaves, except for the efficiency conversion of digesta to cocoon (P < 0.01). Further RNA-Seq analysis revealed 386 differentially transcribed genes between the 2 groups, with 242 upregulated and 144 downregulated genes. GO enrichment analysis showed that differential transcriptional genes were mainly enriched in organic acid metabolism, oxidation-reduction, and drug catabolism. KEGG enrichment analysis showed that differential transcriptional genes were mainly enriched in genetic information processing and metabolism pathways. Our findings provide new insights into the silk secretion and can serve as a reference for future research and application of silkworms fed with artificial diets.

Thermal Stability Enhancement of L-Asparaginase from Corynebacterium glutamicum Based on a Semi-Rational Design and Its Effect on Acrylamide Mitigation Capacity in Biscuits.

Acrylamide is present in thermally processed foods, and it possesses toxic and carcinogenic properties. L-asparaginases could effectively regulate the formation of acrylamide at the source. However, current L-asparaginases have drawbacks such as poor thermal stability, low catalytic activity, and poor substrate specificity, thereby restricting their utility in the food industry. To address this issue, this study employed consensus design to predict the crucial residues influencing the thermal stability of Corynebacterium glutamicum L-asparaginase (CgASNase). Subsequently, a combination of site-point saturating mutation and combinatorial mutation techniques was applied to generate the double-mutant enzyme L42T/S213N. Remarkably, L42T/S213N displayed significantly enhanced thermal stability without a substantial impact on its enzymatic activity. Notably, its half-life at 40 °C reached an impressive 13.29 ± 0.91 min, surpassing that of CgASNase (3.24 ± 0.23 min). Moreover, the enhanced thermal stability of L42T/S213N can be attributed to an increased positive surface charge and a more symmetrical positive potential, as revealed by three-dimensional structural simulations and structure comparison analyses. To assess the impact of L42T/S213N on acrylamide removal in biscuits, the optimal treatment conditions for acrylamide removal were determined through a combination of one-way and orthogonal tests, with an enzyme dosage of 300 IU/kg flour, an enzyme reaction temperature of 40 °C, and an enzyme reaction time of 30 min. Under these conditions, compared to the control (464.74 ± 6.68 µg/kg), the acrylamide reduction in double-mutant-enzyme-treated biscuits was 85.31%, while the reduction in wild-type-treated biscuits was 68.78%. These results suggest that L42T/S213N is a promising candidate for industrial applications of L-asparaginase.

The Role of Chitooligosaccharidolytic ß-N-Acetylglucosamindase in the Molting and Wing Development of the Silkworm Bombyx mori.

The insect glycoside hydrolase family 20 ß-N-acetylhexosaminidases (HEXs) are key enzymes involved in chitin degradation. In this study, nine HEX genes in Bombyx mori were identified by genome-wide analysis. Bioinformatic analysis based on the transcriptome database indicated that each gene had a distinct expression pattern. qRT-PCR was performed to detect the expression pattern of the chitooligosaccharidolytic ß-N-acetylglucosaminidase (BmChiNAG). BmChiNAG was highly expressed in chitin-rich tissues, such as the epidermis. In the wing disc and epidermis, BmChiNAG has the highest expression level during the wandering stage. CRISPR/Cas9-mediated BmChiNAG deletion was used to study the function. In the BmChiNAG-knockout line, 39.2% of female heterozygotes had small and curly wings. The ultrastructure of a cross-section showed that the lack of BmChiNAG affected the stratification of the wing membrane and the formation of the correct wing vein structure. The molting process of the homozygotes was severely hindered during the larva to pupa transition. Epidermal sections showed that the endocuticle of the pupa was not degraded in the mutant. These results indicate that BmChiNAG is involved in chitin catabolism and plays an important role in the molting and wing development of the silkworm, which highlights the potential of BmChiNAG as a pest control target.

Mitochondrial Genome Architecture and Evolutionary Origin of the Yao Silkworm, a Living Fossil of the Domestic Silkworm Bombyx mori (Lepidoptera: Bombycidae).

The Yao silkworm is a unique silkworm resource producing yellow flat plate silk that has only been reared by the Baiku Yao ethnic group in Nandan County, Guangxi Province, China for a thousand years. Here, we report the mitochondrial genomes (mitogenomes) of five Yao silkworm strains and 10 local Guangxi strains of the domestic silkworm (Bombyx mori) L. (Lepidoptera: Bombycidae), and use the resulting mitogenomes and the available Bombyx mitogenomes to characterize their genome architecture and trace the evolutionary origin of the Yao silkworm. The five Yao silkworm mitogenomes exhibited genome architectures identical to typical set of 37 mitochondrial genes (13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs) and a high level of genome sequence similarity with the domestic silkworm. Mitogenome-based phylogenetic reconstruction provided solid evidence that the Yao silkworm shares a common ancestor with the domestic silkworm. Sliding window analysis uncovered a distinct variation pattern in the mitogenome between the Yao silkworm and the other domestic silkworm strains. The phylogenetic analyses revealed a basal placement of the Yao silkworm among all available domestic silkworm strains, indicating that the Yao silkworm is an ancient population of the domestic silkworm. Our data indicated that the Yao silkworm (B. mori) is a lineage of the domestic silkworm, which for the first time provides insights into the origin of the Yao silkworm.

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.

Quadruple-responsive nanoparticle-mediated targeted combination chemotherapy for metastatic breast cancer.

The synergism of combination chemotherapy can only be achieved under specific drug ratios. Herein, hyaluronic acid (HA)-functionalized regenerated silk fibroin-based nanoparticles (NPs) were used to concurrently deliver curcumin (CUR) and 5-fluorouracil (5-FU) at various weight ratios (3.3 : 1, 1.6 : 1, 1.1 : 1, 1 : 1, and 1 : 1.2) to breast tumor cells. The generated HA-CUR/5-FU-NPs were found to have desirable particle sizes (around 200 nm), narrow size distributions, and negative zeta potentials (about -26.0 mV). Interestingly, these NPs showed accelerated drug release rates when they were exposed to buffers that mimicked the multi-hallmarks in the tumor microenvironment (pH/hydrogen peroxide/glutathione/hyaluronidase). The surface functionalization of NPs with HA endowed them with in vitro and in vivo breast tumor-targeting properties. Furthermore, we found that the co-loading of CUR and 5-FU in HA-functionalized NPs exhibited obvious synergistic anti-cancer, pro-apoptotic, and anti-migration effects, and the strongest synergism was found at the CUR/5-FU weight ratio of 1 : 1.2. Most importantly, mice experiments revealed that HA-CUR/5-FU-NPs (1 : 1.2) showed a superior anti-cancer activity against metastatic breast cancer compared to the single drug-loaded NPs and non-functionalized CUR/5-FU-NPs (1 : 1.2). Collectively, these results demonstrate that HA-CUR/5-FU-NPs (1 : 1.2) can be exploited as a robust nanococktail for the treatment of breast cancer and its lung metastasis.

New insight into the mechanism of in vivo fibroin self-assembly and secretion in the silkworm, Bombyx mori.

Fibroin of the silkworm consists of fibroin heavy chain (Fib-H) with hydrophobic intermediate repeats flanked by hydrophilic N and C terminal domains (NTD and CTD, respectively), fibroin light chain (Fib-L), and P25. However, the respective roles of each polypeptide in silk processing remain largely unknown. Here, a series of transgenic silkworms with different fusion gene expression cassettes were created in order to selectively express different fluorescent fusion proteins in silk glands. The roles of different components in silk processing were investigated via observing and analyzing the movement and distribution of these proteins in the silk gland and in cocoon silk. The data showed that hydrophilic NTDs were distributed on the surface of micelles, providing sufficient electrostatic repulsion to prevent premature crystallization of silk proteins. Hydrophilic CTD==Ls ("==" represents the disulfide bond) were located on the inner layer of micelles to control the solubility of large micelles. The results presented here elucidated the underlying mechanisms of silkworm silk processing in vivo. This is significant for the development of artificial spinning technology, novel silk biomaterials, and silk gland expression systems.

The complete mitochondrial genome of Yao silkworm (Bombyx mori).

Here, we describe the first complete mitochondrial genome of Yao silkworm, a unique silkworm resource native at Guangxi, China. This circular molecule is 15,656 bp long and contains the typical set of 37 genes (13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes) and one non-coding A + T-rich region of 494 bp long. The genome organization and gene arrangement are identical to those observed in all available Bombyx mori strains. The phylogenetic tree inferred from Bayesian inference provides a molecular evidence that Yao silkworm belongs to the domestic silkworm (B. mori), rather than a novel silkworm species.

XPC epigenetic silence coupled with p53 alteration has a significant impact on bladder cancer outcome.

PURPOSE: Varied XPC genetics are related to bladder cancer susceptibility. We determined whether decreased XPC expression influences bladder cancer malignancy and clinical outcome. MATERIALS AND METHODS: Changes in XPC and p53 expression were detected by immunochemistry in 108 bladder cancers, including 29 papillary neoplasms of low malignant potential, and 48 low and 31 high grade lesions, of which 47 were stage Ta-T1 and 61 were stage T2-T3. XPC mRNA and methylation were evaluated in fresh tissue by real-time reverse transcriptase and methylation specific polymerase chain reaction. The clinical value of altered XPC and p53 expression was analyzed in 66 bladder cancers, including 6 papillary neoplasms of low malignant potential, and 41 low and 19 high stage lesions, of which 26 were stage Ta-T1 and 40 were stage T2-T3, by the Kaplan-Meier method and Cox proportional hazards regression. RESULTS: The XPC defect was associated with bladder cancer higher pathological grade, metastasis and p53 mutation. Patients with XPC(-)/p53(+) had shorter survival than those with bladder cancer without XPC(-)/p53(+) (p = 0.0127). Cox regression analysis showed that XPC expression may be a potential predictive factor for bladder cancer (p = 0.043). In bladder cancer xpc gene hypermethylation was significantly higher than in normal mucosa (p = 0.0437). CONCLUSIONS: Lower mRNA may be the result of XPC hypermethylation in bladder cancer. Epigenetic defects in the XPC gene impact bladder cancer malignant behavior and may also predict poor outcome in some bladder cancer cases, as characterized by p53 pathway alteration.