Integrated Analysis of Machine Learning and Deep Learning in Silkworm Pupae (Bombyx mori) Species and Sex Identification.

Hybrid pairing of the corresponding silkworm species is a pivotal link in sericulture, ensuring egg quality and directly influencing silk quantity and quality. Considering the potential of image recognition and the impact of varying pupal postures, this study used machine learning and deep learning for global modeling to identify pupae species and sex separately or simultaneously. The performance of traditional feature-based approaches, deep learning feature-based approaches, and their fusion approaches were compared. First, 3600 images of the back, abdomen, and side postures of 5 species of male and female pupae were captured. Next, six traditional descriptors, including the histogram of oriented gradients (HOG), and six deep learning descriptors, including ConvNeXt-S, were utilized to extract significant species and sex features. Finally, classification models were constructed using the multilayer perceptron (MLP), support vector machine, and random forest. The results indicate that the {HOG + ConvNeXt-S + MLP} model excelled, achieving 99.09% accuracy for separate species and sex recognition and 98.40% for simultaneous recognition, with precision-recall and receiver operating characteristic curves ranging from 0.984 to 1.0 and 0.996 to 1.0, respectively. In conclusion, it can capture subtle distinctions between pupal species and sexes and shows promise for extensive application in sericulture.

Probiotic Bacillus subtilis contributes to the modulation of gut microbiota and blood metabolic profile of hosts.

Probiotic Bacillus subtilis has beneficial efficacy on host's health. The microbiota-gut-blood system (MGBS) plays a crucial role in maintaining the homeostasis of hosts. However, the mechanism by which the probiotic B. subtilis positively acts on the MGBS of hosts remains unclear. Herein, we used an interspecies animal model to explore the causal associations between this bacterium and the micro-ecology balance and circulatory homeostasis of hosts. Results showed that the body weight of hosts significantly increased after probiotic B. subtilis supplementation (P < 0.05). Enterococcus was found to be the most important microbial marker causing the intergroup differences observed herein, and its relative abundance remarkably increased after B. subtilis supplementation. In addition, the supplementation of B. subtilis induced significant alterations in the levels of circulating metabolites, such as serine, arginine, adenine, uric acid, and pyridoxal (P < 0.05), indicating that B. subtilis modulated the metabolic profile of blood circulation in the host. The metabolisms of amino acids, purine, and vitamin B were the primary pathways modulated by B. subtilis. In conclusion, probiotic B. subtilis substantially introduced subtle but positive changes in the host's gut microbiome, and it promoted the physiological activity of the host by modulating circulating metabolites. The study provides a theoretical reference for the application of probiotic B. subtilis to improve the health state of specific populations.

PITPNA-AS1/miR-98-5p to Mediate the Cisplatin Resistance of Gastric Cancer.

Gastric cancer (GC) is the most deadly gastrointestinal malignancy with high incidence and mortality. Although, molecular mechanisms which drive gastric cancer progression are extensively investigated, the roles of long noncoding RNA (lncRNA) in gastric cancer growth and drug sensitivity remain unclear. Platinum is a mainstay to treat gastric cancer, and platinum resistance always leads to the local recurrence of gastric cancer. Therefore, it is important to identify biomarkers or therapeutic targets to sensitize gastric cancer to platinum. In this study, we employ noncoding RNA sequencing and found that lncRNA PITPNA-AS1 is overexpressed in gastric cancer tissues and associated with poor survival of gastric cancer patients. Kockdown of PITPNA-AS1 in gastric cancer cells significantly inhibited cell growth and triggered apoptotic cell death in gastric cancer cells. Also, cisplatin treatment could decrease PITPNA-AS1 levels in gastric cancer cells through inhibiting H3K27ac. Besides, PITPNA-AS1 is elevated in cisplatin-resistant gastric cancer cells and tissues, PITPNA-AS1 knockdown could sensitize gastric cancer cells to cisplatin treatment. Furthermore, we identified that PITPNA-AS1 directly interacts and inhibits miR-98-5p. Therefore, PITPNA-AS1 could be served as a potential biomarkers and curative therapeutic targets for gastric cancer progression.

Unpredictable recombination of PB transposon in Silkworm: a potential risk.

The piggyBac (PB) transposon is the most widely used vector for generating transgenic silkworms. The stability of the PB transposon in the receptor is a serious concern that requires attention because of biosafety concerns. In this study, we found that the transgene silkworm developed loss of reporter gene traits. To further investigate the regularity, we traced the genes and traits of this silkworm. After successful alteration of the silkworm genome with the MASP1 gene (named red-eyed silkworm; RES), silkworm individuals with lost reporter genes were found after long-term transgenerational breeding and were designated as the white-eyed silkworm (WES). PCR amplification indicated that exogenous genes had been lost in the WES. Testing was conducted on the PB transposons, and the left arm (L arm) did not exist; however, the right arm (R arm) was preserved. Amino acid analysis showed that the amino acid content of the WES changed versus the common silkworm and RES. These results indicate that the migration of PB transposons in Bombyx mori does occur and is unpredictable. This is because the silkworm genome contains multiple PB-like sequences that might influence the genetic stability of transgenic lines. When using PB transposons as a transgene vector, it is necessary to fully evaluate and take necessary measures to prevent its re-migration in the recipient organism. Further experiments are needed if we want to clarify the regularity of the retransposition phenomenon and the direct and clear association with similar sequences of transposons.

Ultrasound assisted surface micro-dissolution to embed nano TiO2 on cotton fabrics in ZnCl2 aqueous solution.

In this paper, a simple and environment friendly approach was used to prepare the multifunctional composite fabrics via coating commercial TiO2 nanoparticles (NPs) on the surface of cotton fibers by surface micro-dissolution process in 55%wt ZnCl2 aqueous solution under the aid of ultrasound without any adhesive. The TiO2/cotton composite fabrics were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and thermal gravity (TG) methods. The treated fabric had photocatalytic property in Rhodamine B (RhB) degradation, and had good antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) cultures. The results of contamination degradation and antibacterial experiments confirmed that ultrasonic vibration had a significant effect on the tightly coating of nanoparticles on cotton fiber surface in the process of micro-dissolution. Even after intense ultrasonic washing, the contamination degradation rate and antibacterial efficiency of S3 (with ultrasonic) still reach 95% and 99% respectively, while S4 (without ultrasonic) only remained 72% and 59% of S2, which was attributed that ultrasonic facilitated the entry and load of nanoparticles onto the fabrics. The mechanical property of treated cotton fabrics kept well.

A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

Analysis of a new type of major ampullate spider silk gene, MaSp1s.

Spider dragline silk, which is secreted from the major ampullate silk glands, is a unique fibrous protein with a combination of tensile strength and elasticity. Here, we describe a new short type of dragline silk gene, Cyrtophora moluccensis MaSp1s. The full-length gene is only 1320 base pairs (bp), which encodes 439 amino acids that includes the intact non-repetitive N-terminal (149 residues), C-terminal (98 residues) and so-called repetitive regions (192 residues); the deduced molecular weight is approximately 40 kDa. The sequence analysis demonstrated that the two termini are highly homologous to the other characterized dragline silk genes but that the so-called repetitive region is different. Our results suggest that MaSp1s is a possible new characteristic dragline gene; the discovery of this gene should enhance our understanding of the major ampullate spider silk genes.

Characterization of a cysteine-rich protein specifically expressed in the silk gland of caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

A novel protein, Smsp-72k, was found to be selectively expressed in the silk gland of aquatic larvae of the Stenopsychid caddisfly (Stenopsyche marmorata). The protein was characterized by an abundance of cysteine (13.97%) and charged residues (47.21%). Amino acids with hydroxyl side-chains accounted for an additional 10% of the Smsp-72k protein, with serine at 4.4% and threonine at 5.6%. A cysteine-rich repetitive sequence is common to many potential and known underwater adhesive/cement proteins and cell-cell adhesion molecules. We hypothesized that Smsp-72k is an adhesive/cement protein that increases the adhesiveness of the silk fiber of S. marmorata. The hydroxyl groups of Smsp-72k might form a link with the heavy chain fibroin of S. marmorata, removing the weak boundary-water layer and allowing the spreading of the silk protein onto the surface of the substratum during the process of adhesion.

Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

The silks of both Lepidoptera and its sister order Trichoptera contain a homologue of heavy chain (H-fibroin), which is assumed to determine the physical properties of the fiber, such as elasticity and toughness. The long repetitive region of the H-fibroin caddisfly Stenopsyche marmorata shows a conspicuous hierarchical structure that is composed of huge units, which are mainly constructed from four large blocks (SA, SB, SC and SD) arranged in an orderly fashion. Each block contains short, distinct motifs such as SXSXSX(SX), GPXG(X)(1-3) or triplet GGX, which also occur in lepidopteran and spider filaments. The SA, SB and SC blocks have nearly fixed amino acid numbers, while the length of the SD block varies, usually due to a variable number of GPXGXXX repeats. The multiple sandwich structure that occurs in the SB block is assumed to be unique to the caddisfly and may be related to the use of silk in an aqueous environment. The overall average of hydrophilicity in the repetitive H-fibroin region of S. marmorata is -0.609, whereas hydrophobicity prevails in most lepidopteran H-fibroins. Gly (29.51%), Pro (11.28%) and Ser (10.90%) are the three predominant amino acids of H-fibroin, and the high content of essential amino acids reflects the energy-rich food resources of the caddisfly. The H-fibroin of S. marmorata is about 400-500 kDa and expressed in both the middle and posterior silk glands, which is different from the expression pattern in Lepidoptera species.

New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.

We constructed three different fibroin H-chain expression systems to estimate the efficacy of producing recombinant proteins in the cocoon of transgenic silkworms. The results showed that the three different EGFP/H-chain fusion genes were all expressed selectively in the posterior silk gland of the transgenic silkworm. The recombinant protein content of transgenic silkworm cocoons is up to 15% (w/w) when using the most highly efficient H-chain expression system. To our knowledge, in comparison with silkworm silk gland expression systems in the literature, the highly efficient expression system developed in this study is the most efficient silkworm silk gland expression system to date. This expression system is the best candidate for foreign gene production and for creation of novel functional silk material. The results suggested the N-terminal domain and the intron of the H-chain gene are important in the secretion of fibroin and its transcription, respectively.

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons.

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.

Novel molecular and mechanical properties of egg case silk from wasp spider, Argiope bruennichi.

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have various mechanical properties. To date, the fibroin sequences encoding egg case fibers have not been fully determined. To gain further understanding of a recently reported spider silk protein gene family, several novel strategies were utilized in this study to isolate two full-length cDNAs of egg case silk proteins, cylindrical silk protein 1 (CySp1, 9.1 kb) and cylindrical silk protein 2 (CySp2, 9.8 kb), from the wasp spider, Argiope bruennichi. Northern blotting analysis demonstrated that CySp1 and CySp2 are selectively expressed in the cylindrical glands. The amino acid composition of raw egg case silk was closely consistent with the deduced amino acid composition based on the sequences of CySp1 and CySp2, which supports the assertion that CySp1 and CySp2 represent two major components of egg case silk. CySp1 and CySp2 are primarily composed of remarkable homogeneous assemble repeats that are 180 residues in length and consist of several complex subrepeats, and they contain highly homologous C-termini and markedly different N-termini. Our results suggest a possible link between CySp1 and CySp2. In addition, comparisons of stress/strain curves for dragline and egg case silk from Argiope bruennichi showed obvious differences in ultimate strength and extensibility, and similarities in toughness.

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Unique molecular architecture of egg case silk protein in a spider, Nephila clavata.

We describe a unique silk protein secreted from the cylindrical silk glands of the spider Nephila clavata. This silk is primarily composed of three proteins, whose transcripts of approximately 16.0, 14.5 and 13.0 kb are homologous to one another in two termini and repetitive units, as determined on Northern blotting. Its overall organization shows that it is similar to other characterized silk proteins, including in the mainly central repetitive region as well as the non-repetitive N-terminal (166 residues) and C-terminal (176 residues) parts. However, up to 90% of the protein consists of highly ordered repetitive structures that are not found in other silks. The repetitive region mainly consists of several types of complexes and remarkably conserved polypeptide repeats. The assembled repeat units (A1B1) contain a high proportion of Ala (30.41%), Ser (25.15%), and residues with hydrophobic side chains (22.22% for Gly, Leu, Ile, Val and Phe combined). The presence of Ser-rich and GVGAGASA motifs suggests the formation of a beta-sheet. The repetitive region is characterized by alternating arrays of hydrophobic and hydrophilic blocks. The results suggested that this egg case silk is an exceptional protein when compared with previously investigated spider silks.