The acetyltransferase BmCBP changes the acetylation modification of BmSP3 and affects its protein expression in silkworm, Bombyx mori.

BACKGROUND: Protein acetylation is an important post-translational modification (PTM) that widely exists in organisms. As a reversible PTM, acetylation modification can regulate the function of proteins with high efficiency. In the previous study, the acetylation sites of silkworm proteins were identified on a large scale by nano-HPLC/MS/MS (nanoscale high performance liquid chromatography-tandem secondary mass spectrometry), and a total of 11 acetylation sites were discovered on Bombyx mori nutrient-storage protein SP3 (BmSP3). The purpose of this study was to investigate the effect of acetylation level on BmSP3. METHODS AND RESULTS: In this study, the acetylation of BmSP3 was further verified by immunoprecipitation (IP) and Western blotting. Then, it was confirmed that acetylation could up-regulate the expression of BmSP3 by improving its protein stability in BmN cells. Co-IP and RNAi experiments showed acetyltransferase BmCBP could bind to BmSP3 and catalyze its acetylation modification, then regulate the expression of BmSP3. Furthermore, the knock-down of BmCBP could improve the ubiquitination level of BmSP3. Both acetylation and ubiquitination occur on the side chain of lysine residues, therefore, we speculated that the acetylation of BmSP3 catalyzed by BmCBP could competitively inhibit its ubiquitination modification and improve its protein stability by inhibiting ubiquitin-mediated proteasome degradation pathway, and thereby increase the expression and intracellular accumulation. CONCLUSIONS: BmCBP catalyzes the acetylation of BmSP3 and may improve the stability of BmSP3 by competitive ubiquitination. This conclusion provides a new functional basis for the extensive involvement of acetylation in the regulation of nutrient storage and utilization in silkworm, Bombyx mori.

Gastrodia elata specific miRNA attenuates neuroinflammation via modulating NF-κB signaling pathway.

AIMS: Based on our previous research on the specific miRNAs identified from Gastrodia elata, we selected Gas-miR2-3p to investigate its effects on neuroinflammation via in vitro and in vivo experiments. RESULTS: RT-qPCR analysis indicated that G. elata specific Gas-miR2-3p was detected in all murine tissues post-oral administration, suggesting their potential as orally bioavailable miRNA. The analysis of RT-qPCR, Western blotting and ELISA assays consistently demonstrate that the expression of inflammatory factors as TNF-α, IL-6, IL-1ß was decreased and the expression levels of p-p65 and p-IκBα were downregulated after the action of Gas-miR2-3p in both cell and animal experiments. CONCLUSION: Gas-miR2-3p can attenuate neuroinflammation by regulating the inflammation factors and suppressing the activation of the NF-κB signaling pathway. Our findings indicate that G. elata miRNAs, as novel active components, perform a modulatory role in the NF-κB signaling pathway associated with neuroinflammation in a cross-species way.

Biochemical Characterization and Functional Analysis of Glucose Regulated Protein 78 from the Silkworm Bombyx mori.

The glucose regulated protein (GRP78) is an important chaperone for various environmental and physiological stimulations. Despite the importance of GRP78 in cell survival and tumor progression, the information regarding GRP78 in silkworm Bombyx mori L. is poorly explored. We previously identified that GRP78 expression was significantly upregulated in the silkworm Nd mutation proteome database. Herein, we characterized the GRP78 protein from silkworm B. mori (hereafter, BmGRP78). The identified BmGRP78 protein encoded a 658 amino acid residues protein with a predicted molecular weight of approximately 73 kDa and comprised of two structural domains, a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). BmGRP78 was ubiquitously expressed in all examined tissues and developmental stages by quantitative RT-PCR and Western blotting analysis. The purified recombinant BmGRP78 (rBmGRP78) exhibited ATPase activity and could inhibit the aggregating thermolabile model substrates. Heat-induction or Pb/Hg-exposure strongly stimulated the upregulation expression at the translation levels of BmGRP78 in BmN cells, whereas no significant change resulting from BmNPV infection was found. Additionally, heat, Pb, Hg, and BmNPV exposure resulted in the translocation of BmGRP78 into the nucleus. These results lay a foundation for the future identification of the molecular mechanisms related to GRP78 in silkworms.

Neuroprotection and Mechanism of Gas-miR36-5p from Gastrodia elata in an Alzheimer's Disease Model by Regulating Glycogen Synthase Kinase-3ß.

Alzheimer's disease (AD) is currently the most common neurodegenerative disease. Glycogen synthase kinase 3ß (GSK-3ß) is a pivotal factor in AD pathogenesis. Recent research has demonstrated that plant miRNAs exert cross-kingdom regulation on the target genes in animals. Gastrodia elata (G. elata) is a valuable traditional Chinese medicine that has significant pharmacological activity against diseases of the central nervous system (CNS). Our previous studies have indicated that G. elata-specific miRNA plays a cross-kingdom regulatory role for the NF-κB signaling pathway in mice. In this study, further bioinformatics analysis suggested that Gas-miR36-5p targets GSK-3ß. Through western blot, RT-qPCR, and assessments of T-AOC, SOD, and MDA levels, Gas-miR36-5p demonstrated its neuroprotective effects in an AD cell model. Furthermore, Gas-miR36-5p was detected in the murine brain tissues. The results of the Morris water maze test and western blot analysis provided positive evidence for reversing the learning deficits and hyperphosphorylation of Tau in AD mice, elucidating significant neuroprotective effects in an AD model following G. elata RNA administration. Our research emphasizes Gas-miR36-5p as a novel G. elata-specific miRNA with neuroprotective properties in Alzheimer's disease by targeting GSK-3ß. Consequently, our findings provide valuable insights into the cross-kingdom regulatory mechanisms underlying G. elata-specific miRNA, presenting a novel perspective for the treatment of Alzheimer's disease.

The acetylation modification regulates the stability of Bm30K-15 protein and its mechanism in silkworm, Bombyx mori.

The 30 K proteins are the major silkworm hemolymph proteins and are involved in a variety of physiological processes, such as nutrient and energy storage, embryogenesis, immune response, and inhibition of apoptosis. The Bm30K-15 protein is one of the 30 K proteins and is abundant in the hemolymph of fifth instar silkworm larva. We previously found that the Bm30K-15 protein can be acetylated. In the present study, we found that acetylation can improve the protein stability of Bm30K-15. Further exploration confirmed that the increase in protein stability by acetylation was caused by competition between acetylation and ubiquitination. In summary, these findings aim to provide insight into the effect of acetylation modification on the protein level and stability of the Bm30K-15 and the possible molecular mechanism of its existence in silkworm, Bombyx mori.

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm, Bombyx mori.

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H2 O2 . In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H2 O2 -induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.

Cloning, expression profiling, and acetylation identification of alpha-tubulin N-acetyltransferase 1 from Bombyx mori.

Alpha-tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α-tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth-instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth-instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time-dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site-specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself.

Transposable Element Bm1645 is a Source of BmAGO2-associated Small RNAs that affect its expression in Bombyx mori.

BACKGROUND: A transposable element (TE) is a DNA fragment that can change its position within a genome. Transposable elements play important roles in maintaining the stability and diversity of organisms by transposition. Recent studies have shown that approximately half of the genes in Bombyx mori are TEs. RESULTS: We systematically identified and analyzed the BmAGO2-associated TEs, which exceed 100 in the B. mori genome. Additionally, we also mapped the small RNAs associated with BmAGO2 in B.mori. The transposon Bm1645 is the most abundant TE associated with BmAGO2, and Bm1645-derived small RNAs represent a small RNA pool. We determined the expression patterns of several Bm1645-derived small RNAs by northern blotting, and the results showed there was differential expression of multiple small RNAs in normal and BmNPV-infected BmN cells and silkworms from various developmental stages. We confirmed that four TE-siRNAs could bind to BmAGO2 using EMSA and also validated the recognition sites of these four TE-siRNAs in Bm1645 by dual-luciferase reporter assays. Furthermore, qRT-PCR analysis revealed the overexpression of the four TE-siRNAs could downregulate the expression of Bm1645 in BmN cells, and the transcription of Bm1645 was upregulated by the downregulation of BmAGO2. CONCLUSIONS: Our results suggest Bm1645 functions as a source of small RNAs pool and this pool can produce many BmAGO2-associated small RNAs that regulate TE's expression.

Different nitrogen sources change the transcriptome of welan gum-producing strain Sphingomonas sp. ATCC 31555.

To reveal effects of different nitrogen sources on the expressions and functions of genes in Sphingomonas sp. ATCC 31555, it was cultivated in medium containing inorganic nitrogen (IN), organic nitrogen (ON), or inorganic-organic combined nitrogen (CN). Welan gum production and bacterial biomass were determined, and RNA sequencing (RNA-seq) was performed. Differentially expressed genes (DEGs) between the different ATCC 31555 groups were identified, and their functions were analyzed. Welan gum production and bacterial biomass were significantly higher in the ON and CN groups compared with those in the IN group. RNA-seq produced 660 unigenes, among which 488, 731, and 844 DEGs were identified between the IN vs. ON, IN vs. CN, and ON vs. CN groups, respectively. All the DEGs were related significantly to metabolic process and signal transduction. DEGs between the IN vs. CN and ON vs. CN groups were potentially associated with bacterial chemotaxis. Real-time PCR confirmed the expressions of selected DEGs. Organic nitrogen led to higher bacterial biomass and welan gum production than inorganic nitrogen, which might reflect differences in gene expression associated with metabolic process, signal transduction, and bacterial chemotaxis induced by different nitrogen sources.

Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity.

Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection.

Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects.

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae.

Recent studies have demonstrated that recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) produced by the silkworm pupae bioreactor is absorbed into blood through oral administration and functions as an active cytokine. The aim of this study was to further examine and identify synergetic protein factors in silkworm pupae that improve rhGM-CSF absorption via an oral route. The concentrations of rhGM-CSF in serum were evaluated in mice after oral administration of rhGM-CSF using different chemical compositions of silkworm pupae as pharmaceutical excipients. The experimental data revealed that the supernatant lyophilized powder (SLP) of a homogenized slurry of silkworm pupae caused a significant increase in the rhGM-CSF level in blood when rhGM-CSF was orally administered with SLP, suggesting that synergetic protein factors that improve the oral absorption of rhGM-CSF primarily exist in SLP. As shown by scanning electron microscopy, microspheres were formed when rhGM-CSF was coated with SLP. Animal experimental data showed that the absorption of orally administered rhGM-CSF through the gastrointestinal (GI) tract primarily resulted from protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration. Surface plasmon resonance spectroscopy analysis demonstrated that several protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration were bound to rhGM-CSF. Proteins bound to rhGM-CSF by liquid chromatography-mass spectrometry were identified as chymotrypsin inhibitor SCI-II precursor, cationic peptide CP8 precursor, Kazal-type proteinase inhibitor, and chymotrypsin inhibitor SCI-I. These findings indicate that these proteinase inhibitors play an important role in improving rhGM-CSF absorption in the GI tract.

A New Member of the TBC1D15 Family from Chiloscyllium plagiosum: Rab GTPase-Activating Protein Based on Rab7 as a Substrate.

APSL (active peptide from shark liver) is a hepatic stimulator cytokine from the liver of Chiloscyllium. It can effectively protect islet cells and improve complications in mice with alloxan-induced diabetes. Here, we demonstrate that the APSL sequence is present in the N-terminus of novel TBC (Tre-2, Bub2 and Cdc16) domain family, member 15 (TBC1D15) from Chiloscyllium plagiosum. This shark TBC1D15 gene, which contains an ORF of 2088 bp, was identified from a cDNA library of regenerating shark liver. Bioinformatic analysis showed that the gene is highly homologous to TBC1D15 genes from other species. Moreover, the N-terminus of shark TBC1D15 contains a motif of unknown function (DUF3548), which encompasses the APSL fragment. Rab-GAP activity analysis showed that shark TBC1D15 is a new member of the TBC1D15 family. These results demonstrated that shark TBC1D15 possesses Rab-GAP activity using Rab7 as a substrate, which is a common property of the TBC1D15 family. The involvement of APSL at the N-terminus of TBC1D15 also demonstrates that this protein might be involved in insulin signaling and may be associated with the development of type 2 diabetes. The current findings pave the way for further functional and clinical studies of these proteins from marine sources.

BmHSP20.8 is Localized in the Mitochondria and has a Molecular Chaperone Function In Vitro.

Heat shock proteins (HSPs) are abundant and ubiquitous in almost all organisms from bacteria to mammals. BmHSP20.8 is a small (sHSP) in Bombyx mori that contains a 561 bp open reading frame that encodes a protein of 186 amino acid residues with a predicted molecular mass of 20.8 kDa. The subcellular localization prediction indicated that BmHSP20.8 is likely distributed in the mitochondria with a 51% probability. To identify the subcellular localization of BmHSP20.8, three recombinant vectors were constructed and used to transfect BmN cells. The cytoplasmic and mitochondrial proteins were extracted 72 h after transfection. The Western blot showed that recombinant BmHSP20.8 exists only in the mitochondria. To locate the mitochondrial localization signal domain of BmHSP20.8 more accurately, we cloned four truncated recombinant vectors. The Western blot analysis of the cytoplasmic and mitochondrial proteins showed that the mitochondrial localization signal domain of BmHSP20.8 is located between amino acids 143 to 186. We constructed the pETduet-HIS-SUMO-BmHSP20.8 vector and a soluble BmHSP20.8 was expressed. In a citrate synthase (CS) thermal aggregation experiment, we found that the recombinant BmHSP20.8 protein can protect CS from aggregating at 43 and 48 °C and thus exhibited molecular chaperone activity. Taken together, the results showed that BmHSP20.8 could be a mitochondrial protein and has a molecular chaperone activity, suggesting an important role in mitochondria.

MicroRNA-452 promotes tumorigenesis in hepatocellular carcinoma by targeting cyclin-dependent kinase inhibitor 1B.

Dysregulation of miR-452 has been observed in many tumors, but its biological function in hepatocellular carcinoma (HCC) is still unknown. Our results showed that miR-452 expression is significantly increased in HCC tissues and HCC cell lines. We also found that overexpression of miR-452 dramatically accelerated proliferation, induced cell cycle from G1 to S transition, and blocked apoptosis of HCC cells. Migration and matrigel invasion assays indicated that miR-452 significantly promotes HepG2 and QGY-7703 cells migration and invasion in vitro. Further studies showed that miR-452 directly targets the 3'-untranslated region of cyclin-dependent kinase inhibitor 1B (CDKN1B), ectopic miR-452 expression suppressed CDKN1B expression on mRNA and protein level. Silencing CDKN1B by small interfering RNA resembled the phenotype resulting from ectopic miR-452 expression. This study provides new insights into the potential molecular mechanisms that miRNA-452 contributed to HCC.

Anti-diabetic effects of CTB-APSL fusion protein in type 2 diabetic mice.

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.

Unveiling the impact of cryptic plasmids curing on Escherichia coli Nissle 1917: massive increase in Ag43c expression.

Escherichia coli Nissle 1917 (EcN) is an important chassis strain widely used for the development of live biotherapeutic products (LBPs). EcN strain naturally harbors two cryptic plasmids with unknown function. During the development of LBPs using EcN strain, the cryptic plasmids were cured usually to avoid plasmid incompatibility or alleviate metabolic burdens associated with these cryptic plasmids. While the cryptic plasmids curing in EcN may appear to be a routine procedure, the comprehensive impact of cryptic plasmids curing on the EcN strain remains incompletely understood. In the present study, the effects of cryptic plasmids curing on EcN were investigated using transcriptome sequencing. The results revealed that only a small number of genes showed significant changes in mRNA levels after cryptic plasmid curing (4 upregulated and 6 downregulated genes), primarily involved in amino acid metabolism. Furthermore, the flu gene showed the most significant different expression, encoding Antigen 43 (Ag43) protein, a Cah family adhesin. Mass spectrometry analysis further confirmed the significant increase in Ag43 expression. Ag43 is commonly present in Escherichia coli and mediates the bacterial autoaggregation. However, despite the upregulation of Ag43 expression, no Ag43-mediated cell self-sedimentation was observed in the cured EcN strain. These findings contribute to making informed decisions regarding the curing of the cryptic plasmids when Escherichia coli Nissle 1917 is used as the chassis strain.

Effect of acute exposure of Hg on physiological parameters and transcriptome expression in silkworms (Bombyx mori).

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

RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori.

BACKGROUND: Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. RESULTS: Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. CONCLUSIONS: Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.

A shark liver gene-derived active peptide expressed in the silkworm, Bombyx mori: preliminary studies for oral administration of the recombinant protein.

Active peptide from shark liver (APSL) is a cytokine from Chiloscyllium plagiosum that can stimulate liver regeneration and protects the pancreas. To study the effect of orally administered recombinant APSL (rAPSL) on an animal model of type 2 diabetes mellitus, the APSL gene was cloned, and APSL was expressed in Bombyx mori N cells (BmN cells), silkworm larvae and silkworm pupae using the silkworm baculovirus expression vector system (BEVS). It was demonstrated that rAPSL was able to significantly reduce the blood glucose level in mice with type 2 diabetes induced by streptozotocin. The analysis of paraffin sections of mouse pancreatic tissues revealed that rAPSL could effectively protect mouse islets from streptozotocin-induced lesions. Compared with the powder prepared from normal silkworm pupae, the powder prepared from pupae expressing rAPSL exhibited greater protective effects, and these results suggest that rAPSL has potential uses as an oral drug for the treatment of diabetes mellitus in the future.