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Abstract Background Accompanying islet α- and β-cell dysregulation in type 2 diabetes (T2D) at the microscopic scale, alterations in body composition at the macroscopic scale may affect the pathogenesis of T2D. However, the connections between body composition and islet α-cell and β-cell functions in T2D have not been thoroughly explored. Methods For this cross-sectional study, we recruited a total of 729 Chinese Han patients with T2D in a consecutive manner. Dual-energy X-ray absorptiometry (DXA) was used to measure body composition, which included total bone-free mass, total fat and lean mass, trunk fat and lean mass and limb fat and lean mass. Every patient underwent an oral glucose tolerance test to simultaneously detect glucose, C-peptide and glucagon. The indices of islet α-cell function included fasting glucagon levels and the area under the curve of glucagon after a challenge (AUCglucagon), while the indices of β-cell function included the insulin sensitivity index derived from C-peptide (ISIC-peptide) and the area under the curve of C-peptide after a challenge (AUCC-peptide). Results Among all patients, fat mass, especially trunk fat mass, was significantly correlated with ISIC-peptide and AUCC-peptide levels (r = − 0.330 and 0.317, respectively, p

Background: Increased serum cystatin C (CysC) can predict the onset of typ e 2 diabetes (T2D). Meanwhile, impaired pancreatic α- and β-cell functions get involved in the pathophysiological processes of T2D. So this study was to explo re the relationships between serum CysC levels and pancreatic α- and β-cell functions in T2D. Methods: In this cross-sectional observational study, a total of 2634 p atients with T2D were consecutively recruited. Each recruited patient received a serum CysC test and oral glucose tolerance test for synchronous detection of serum C-peptide and plasma glucagon. As components of pancreatic β-cell function, insulin secretion and sensitivity indices were evaluated by C-peptide area under the curve (AUC-C P) and C-peptide-substituted Matsuda’s index (Matsuda-CP), respectively. Fasting glucagon (F-GLA) and post-challenge glucagon calculated by glucagon area under the c urve (AUC-GLA) were used to assess pancreatic α-cell function. These skewed indices and were further natural log-transformed (ln). Results: With quartiles of serum CysC levels ascending, AUC-CP, F-GLA a nd AUC-GLA were increased, while Matsuda-CP was decreased (P for trend

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV.

β-1,3-glucan recognition proteins (βGRPs) as pattern recognition receptors (PRRs) play an important role in recognizing various pathogens and trigger complicated signaling pathways in insects. In this study, we identified a Bombyx mori β-1,3-glucan recognition protein gene named BmβGRP4, which showed differential expression, from a previous transcriptome database. The full-length cDNA sequence was 1244 bp, containing an open reading frame (ORF) of 1128 bp encoding 375 amino acids. BmβGRP4 was strongly expressed in the larval stages and highly expressed in the midgut of B. mori larvae in particular. After BmNPV infection, the expression of BmβGRP4 was reduced significantly in the midgut. Furthermore, a significant increase in the copy number of BmNPV was observed after the knockdown of BmβGRP4 in 5th instar larvae, while the overexpression of BmβGRP4 suppressed the proliferation of BmNPV in BmN cells. Subsequently, the expression analysis of several apoptosis-related genes and observation of the apoptosis morphology demonstrated that overexpression of BmβGRP4 facilitated apoptosis induced by BmNPV in BmN cells. Moreover, BmβGRP4 positively regulated the phosphatase and tensin homolog gene (BmPTEN), while expression of the inhibitor of apoptosis gene (BmIAP) was negatively regulated by BmβGRP4. Hence, we hypothesize that BmNPV infection might suppress BmPTEN and facilitate BmIAP to inhibit cell apoptosis by downregulating the expression of BmβGRP4 to escape host antiviral defense. Taken together, these results show that BmβGRP4 may play a role in B. mori response to BmNPV infection and lay a foundation for studying its functions.

The molecular mechanism of silkworm resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) infection remains unclear. The chaperonin containing t-complex polypeptide 1 (TCP-1) is essential for the folding of tubulin and actin to produce stable and functional competent protein conformation. However, little is known about this protein in silkworm. In the present study, a gene encoding the TCP-1β protein in silkworm was characterized, which has an open reading fragment of 1,611 bp encoding a predicted 536 amino acid residue-protein with a molecular weight of approximately 57.6 kDa containing a Cpn60_TCP1 functional domain. The sequence conservation is 81.52%. The highest level of BmTCP-1β mRNA expression was found in the midgut, while the lowest was in the hemolymph. To further study the function of BmTCP-1β, expression was knocked down with siRNA in vitro, resulting in significant downregulation of the selected cytoskeletal-related genes, actin and tubulin, which was also confirmed by overexpression of BmTCP-1β in BmN cells using the pIZT/V5-His-mCherry insect vector. Moreover, knockdown of BmTCP-1β significantly prolonged the infection process of BmNPV in BmN cells, which was also verified by overexpression of BmTCP-1β in BmN cells. Based on the results of the present study, we concluded that BmTCP-1β plays a vital role in BmNPV infection by regulating the expression of tubulin and actin. Taken together, our work provides valuable data for the clarification of the molecular mechanism of silkworm resistance to BmNPV infection.

Previous studies have revealed that some proteins in Bombyx mori larvae digestive juice show antiviral activity. Here, based on the label-free proteomics data, BmLipase member H-A (BmLHA) was identified as being involved in the response to BmNPV infection in B. mori larvae digestive juice. In the present study, a gene encoding the BmLHA protein in B. mori was characterized. The protein has an open reading fragment of 999 bp, encoding a predicted 332 amino acid residue-protein with a molecular weight of approximately 35.9 kDa. The phylogenetic analysis revealed that BmLHA shares a close genetic distance with Papilio xuthus Lipase member H-A. BmLHA was highly expressed in the middle part of the B. mori gut, and the expression level increased with instar rising in larvae. There was higher expression of BmLHA in A35 than in P50 strains, and it was upregulated in both A35 and P50 strains, following BmNPV infection. The expression level of VP39 decreased significantly in appropriate recombinant-BmLHA-treated groups compared with the PBS-treated group in B. mori larvae and BmN cells. Meanwhile, overexpression of BmLHA significantly reduced the infectivity of BmNPV in BmN cells. These results indicated that BmLHA did not have digestive function but had anti-BmNPV activity. Taken together, our work provides valuable data for the clarification of the molecular characterization BmLHA and supplements research on proteins of anti-BmNPV activity in B. mori.

Samia cynthia ricini (Lepidoptera: Saturniidae) is an important commercial silk-producing insect; however, in contrast to the silkworm, mulberry leaves are toxic to this insect because the leaves contain the component 1-deoxynojirimycin (DNJ). A transcriptomic analysis of eri-silkworm haemolymph was conducted to examine the genes related to different metabolic pathways and to elucidate the molecular mechanism underlying eri-silkworm haemolymph responses to DNJ. Eight hundred sixty-five differentially expressed genes (DEGs) were identified, among which 577 DEGs were up-regulated and 288 DEGs were down-regulated in the 2% DNJ group compared to control (ddH2O) after 12h. Based on the results of the functional analysis, these DEGs were associated with ribosomes, glycolysis, N-glycan biosynthesis, and oxidative phosphorylation. In particular, according to the KEGG analysis, 138 DEGs were involved in energy metabolism, glycometabolism and lipid metabolism, and the changes in the expression of nine DEGs were confirmed by reverse transcription quantitative PCR (RT-qPCR). Thus, DNJ induced significant metabolic alterations in eri-silkworm haemolymph. These results will lay the foundation for research into the toxic effects of DNJ on eri-silkworm as a model and provide a reference for the exploitation of new drugs in humans.

Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well distributed superfamily in invertebrates, participating in immune mechanisms and other important physiological processes. Here, we investigated the Bmserpin2 gene which was identified from a transcriptome database in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that Bmserpin2 was expressed in all tissues, with maximum expression in fat body. Upon BmNPV infection, the expression of Bmserpin2 was up-regulated in P50 (susceptible strain) and BC9 (resistant strain) in haemocytes, fat body and the midgut. However, up-regulation was delayed in BC9 (48 or 72 h), in contrast to P50 (24 h), after BmNPV infection. Meanwhile, Bmserpin2 could delay or inhibit melanization in silkworm haemolymph. Significant increased PO activity can be observed in Bmserpin2-depleted haemolymph under NPV infection. Furthermore, the viral genomic DNA copy number was decreased in Bmserpin2-depleted haemolymph. We conclude that Bmserpin2 is an inducible gene which might be involved in the regulation of PPO activation and suppressed melanization, and have a potential role in the innate immune system of B. mori.

1-deoxynojirimycin (DNJ) is a natural D-glucose analogue and has a strong physiological activity in inhibiting α-glucosidase in vivo. The antidiabetic effects of DNJ in mice or other mammals were extensively explored, but the physiological and toxic roles of DNJ in insects was seldom reported. In this study, the biological effects of DNJ were examined in midgut extracts of fourth-instar larvae of Eri silkworm (Samia cynthia ricini, Saturniidae). Based on nuclear magnetic resonance (NMR) metabonomics technology, we analyzed the alterations of glycometabolism, lipids, and energy metabolism pathways in the midgut of S. cynthia ricini caused by DNJ. Pattern recognition analysis (partial least square-discriminant analysis, PLS-DA) showed that four groups of latex, 0.25% DNJ, 0.5% DNJ and the mixture of 0.5% DNJ and latex (1:1) were distinctly different from the control group. Moreover, several metabolic pathways of DNJ-mediated modulation in the midgut were identified. Compared with the control group, alanine, succinate, glutamate, and fumarate concentrations decreased in three groups of 0.5% DNJ, latex, and the mixture, choline levels increased in two DNJ groups, and trehalose levels increased in all experimental groups. Therefore, these results suggest that DNJ modulated lipid metabolism by limiting the hydrolysis pathways of phospholipids metabolism. Additionally, DNJ has a potent negative effect on energy metabolism by inhibiting the hydrolysis of trehalose, glycolysis and the tricarboxylic acid (TCA) cycle. Overall, DNJ, as a single-ingredient, is an efficient substance for modulating lipid metabolism and inhibiting energy metabolism.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens causing severe economic losses in sericulture. However, the molecular mechanism of silkworm resistance to BmNPV remains largely unknown. Here, the recurrent parent P50 (susceptible strain) and the near-isogenic line BC9 (resistance strain) were used in a comparative transcriptome study examining the response to infection with BmNPV. A total of 14,300 unigenes were obtained from two different resistant strains; of these, 869 differentially expressed genes (DEGs) were identified after comparing the four transcriptomes. Many DEGs associated with protein metabolism, cytoskeleton, and apoptosis may be involved in the host response to BmNPV infection. Moreover, some immunity related genes were also altered following BmNPV infection. Specifically, after removing genetic background and individual immune stress response genes, 22 genes were found to be potentially involved in repressing BmNPV infection. These genes were related to transport, virus replication, intracellular innate immune, and apoptosis. Our study provided an overview of the molecular mechanism of silkworm resistance to BmNPV infection and laid a foundation for controlling BmNPV in the future.

We aimed to investigate whether 1-deoxynojirimycin (DNJ) modulates glycometabolism and has toxicity in Eri silkworm (Samia cynthia ricini, Saturniidae). In this paper, hemolymph metabolites were used to explore metabolic changes after oral administration of DNJ or mulberry latex and to characterize the biological function of DNJ at the metabolic and systemic levels. Hemolymph samples were collected from fourth-instar larvae of Eri silkworm and ex-vivo high-resolution 1H nuclear magnetic resonance (NMR) spectra were acquired from the collected hemolymph samples. Then the obtained spectra were analyzed by principal component analysis (PCA) and independent-samples t-test. Metabolic pattern recognition analysis of hemolymph samples indicated that the groups of 0.25% DNJ, latex, and the mixture of 0.5% DNJ and latex (1:1) were significantly different from the control group. Moreover, compared to the control group, the groups of 0.25% DNJ, latex, and the mixture of 0.5% DNJ and latex (1:1) showed the decreased levels of citrate, succinate, fumarate, malate, and glutamine in hemolymph, the groups of 0.25% DNJ and the mixture of 0.5% DNJ and latex (1:1) showed the increased levels of trehalose and lactate. In addition, mulberry leaves exude latex was highly toxic to Eri silkworm because rich unidentified high-molecular-weight factor (s) acted as toxic substances. In our results, latex caused 20 deaths among 50 fourth-instar larvae of Eri silkmoth, but DNJ or the mixture did not caused death. All these results suggest that DNJ has a positive impact on the reverse glycometabolism by modulating glycometabolism and inhibiting glucogenesis and energy metabolism. DNJ is a secure substance as a single-ingredient antidiabetic medicine due to its nontoxicity and bioactivity.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause. [ABSTRACT FROM AUTHOR]

Armadillo (ARM)-repeat protein is involved in many biological processes, including cell-cell adhesion and the Wnt signaling pathway. However, the function of ARM-repeat protein in Bombyx mori has not been completely clarified. In this study, a gene encoding ARM-repeat protein was identified, which has an open reading fragment of 1923 bp, encoding a predicted 641 amino acid residues with a molecular weight of approximately 71.2 kDa. BmARM-like mRNA showed highest expression in hemolymph, significantly high expression levels in 4th instar, moth, and egg, stable high expression levels in the early embryonic development, relatively high expression levels in the 1st day and 5th day of the pupa stage, and highest expression levels in the molting stage. What's more, BmARM-like protein could be detected by immunofluorescence in all analyzed tissues, including head, midgut, hemolymph, fat body, testis and ovary. More importantly, the relatively high expression level of BmARM-like mRNA were observed in BC9 (near-isogenic line) following BmNPV infection as compared to P50 (susceptible strain), which was further validated in the midgut of A35 (resistant strain). The expression levels of BmARM-like protein showed similar trends with transcriptional levels in the midgut following BmNPV infection. Based on the analysis above, we speculated that BmARM-like is involved in response against BmNPV infection. [ABSTRACT FROM AUTHOR]

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host's defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporters belong to a superfamily of genes involved in the transport of specific molecules across lipid membranes, as well as insecticide resistance, present in all living organisms. In this study, we combined the Cnaphalocrocis medinals transcriptome database with a bioinformatics approach to identify four C. medinals ABCs (CmABCs), including CmABCG1, CmABCG4, CmABCC2 and CmABCC3. Tissue expression analysis showed that these genes had a tissue-specific expression pattern. CmABCG1 had significantly higher expression in the haemolymph and head compared to the other tissues. The expression of CmABCG4, CmABCC2 and CmABCC3 was highest in the midgut, followed by expression in the fat body. The developmental stage expression analysis showed that CmABCG1, CmABCG4, CmABCC2 and CmABCC3 were mainly expressed in adults. The transcription of CmABCG1, CmABCG4 and CmABCC2 was significantly induced by chlorpyrifos. Taken together, the results of our study provided useful information for understanding of the detoxification system of C. medinalis. [ABSTRACT FROM AUTHOR]

The rice leaf roller (Cnaphalocrocis medinalis) is one of the most destructive agricultural pests. Due to its migratory behavior, it is difficult to control worldwide. To date, little is known about major genes of C. medinalis involved in chitin metabolism and insecticide detoxification. In order to obtain a comprehensive genome dataset of C. medinalis, we conducted de novo transcriptome sequencing which focused on the major feeding stage of fourth-instar larvae, and our work revealed useful information on chitin metabolism and insecticide detoxification and target genes of C. medinalis. We acquired 29,367,797 Illumina reads and assembled these reads into 63,174 unigenes with an average length of 753 bp. Among these unigenes, 31,810 were annotated against the National Center for Biotechnology Information non-redundant (NCBI nr) protein database, resulting in 24,246, 8669 and 18,176 assigned to Swiss-Prot, clusters of orthologous group (COG), and gene ontology (GO), respectively. We were able to map 10,043 unigenes into 285 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Specifically, 16 genes, including five chitin deacetylases, two chitin synthases, five chitinases and four other related enzymes, were identified to be putatively involved in chitin biosynthesis and degradation, whereas 360 genes, including cytochrome P450s, glutathione S-transferases, esterases, and acetylcholinesterases, were found to be potentially involved in insecticide detoxification or as insecticide targets. The reliability of the transcriptome data was determined by reverse transcription quantitative PCR (RT-qPCR) for the selected genes. Our data serves as a new and valuable sequence resource for genomic studies on C. medinalis. The findings should improve our understanding of C. medinalis genetics and contribute to management of this important agricultural pest. [ABSTRACT FROM AUTHOR]