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Summary: Natural silks are renewable proteins with impressive mechanical properties and biocompatibility that are useful in various fields. However, the cellular and spatial organization of silk-secreting organs remains unclear. Here, we combined single-nucleus and spatially resolved transcriptomics to systematically map the cellular and spatial composition of the silk glands (SGs) of mulberry silkworms late in larval development. This approach allowed us to profile SG cell types and cell state dynamics and identify regulatory networks and cell-cell communication related to efficient silk protein synthesis; key markers were validated via transgenic approaches. Notably, we demonstrated the indispensable role of the ecdysone receptor (ultraspiracle) in regulating endoreplication in SG cells. Our atlas presents the results of spatiotemporal analysis of silk-secreting organ architecture late in larval development; this atlas provides a valuable reference for elucidating the mechanism of efficient silk protein synthesis and developing sustainable products made from natural silk.

Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis, a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species has not been identified. In this study, three high-quality genome assemblies were generated for Morus spp. [including dioecious M. notabilis (male and female) and Morus yunnanensis (female)] with genome sizes of 301–329 Mb and were grouped into six pseudochromosomes. Using a combination of genomic approaches, we found that the putative ancestral karyotype of Morus species was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n = 2x = 12). We also characterized a ∼ 6.2-Mb sex-determining region on chromosome 3. Four potential male-specific genes, a partially duplicated DNA helicase gene (named MSDH) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG1/2/3), were identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry from wild populations in terms of flowering and disease resistance were identified. Our study provides an important genetic resource for sex identification research and molecular breeding in mulberry.

Tong et al. describe a super pangenome assembled from long-read sequences of 545 wild and domesticated silkworms. Naturally selected (diapause, aposemantic coloration) or artificially selected (silk yield and fineness) sets of genes are delineated.

The molecular underpinning of silk-producing organs is not well characterized. Here the authors use single-cell RNA sequencing to build an atlas of the silkworm silk gland and reveal the heterogeneity of silk gland cells.

Mulberry (genus Morus) is an economically important woody plant with an altered ploidy level. The variable number of Morus species recognized by different studies indicates that the genus is in need of revision. In this study, the chloroplast (CP) genomes of 123 Morus varieties were de novo assembled and systematically analyzed. The 123 varieties represented six Morus species, namely, Morus alba, Morus nigra, Morus notabilis, Morus rubra, Morus celtidifolia, and Morus serrata. The Morus CP genome was found to be 158,969~159,548 bp in size with 125 genes, including 81 protein coding, 36 tRNA, and 8 rRNA genes. The 87 out of 123 mulberry accessions were assigned to 14 diverse groups with identical CP genome, which indicated that they are maternally inherited and share 14 common ancestors. Then 50 diverse CP genomes occurred in 123 mulberry accessions for further study. The CP genomes of the Morus genus with a quadripartite structure have two inverted repeat (IR) regions (25,654~25,702 bp) dividing the circular genome into a large single-copy (LSC) region (87,873~88,243 bp) and small single-copy (SSC) region (19,740~19,994 bp). Analysis of the phylogenetic tree constructed using the complete CP genome sequences of Morus revealed a monophyletic genus and that M. alba consisted of two clades, M. alba var. alba and M. alba var. multicaulis. The Japanese cultivated germplasms were derived from M. alba var. multicaulis. We propose that the Morus genus be classified into six species, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. rubra, and M. alba with two subspecies, M. alba var. alba and M. alba var. multicaulis. Our findings provide a valuable resource for the classification, domestication, and breeding improvement of mulberry.

Growth promotion and stress tolerance induced by endophytes have been observed in various plants, but their effects on mulberry regularly suffering flood in the hydro-fluctuation belt are less understood. In the present study, endophytic Klebsiella aerogenes HGG15 was screened out from 28 plant growth promotion (PGP) bacteria as having superior PGP traits in vitro and in planta as well as biosafety for silkworms. K. aerogenes HGG15 could actively colonize into roots of mulberry and subsequently transferred to stems and leaves. The 16S ribosomal RNA (V3–V4 variable regions) amplicon sequencing revealed that exogenous application of K. aerogenes HGG15 altered the bacterial community structures of mulberry roots and stems. Moreover, the genus of Klebsiella was particularly enriched in inoculated mulberry roots and was positively correlated with mulberry development and soil potassium content. Untargeted metabolic profiles uncovered 201 differentially abundant metabolites (DEMs) between inoculated and control mulberry, with lipids and organo-heterocyclic compounds being particularly abundant DEMs. In addition, a high abundance of abiotic stress response factors and promotion growth stimulators such as glycerolipid, sphingolipid, indole, pyridine, and coumarin were observed in inoculated mulberry. Collectively, the knowledge gained from this study sheds light on potential strategies to enhance mulberry growth in hydro-fluctuation belt, and microbiome and metabolite analyses provide new insights into the growth promotion mechanisms used by plant-associated bacteria.

Plant growth-promoting rhizobacteria have been shown to play important roles in maintaining host fitness under periods of abiotic stress, and yet their effect on mulberry trees which regularly suffer drought after flooding in the hydro-fluctuation belt of the Three Gorges Reservoir Region in China remains largely uncharacterized. In the present study, 74 bacterial isolates were obtained from the rhizosphere soil of mulberry after drought stress, including 12 phosphate-solubilizing and 10 indole-3-acetic-acid-producing isolates. Bacillus megaterium HGS7 was selected for further study due to the abundance of traits that might benefit plants. Genomic analysis revealed that strain HGS7 possessed multiple genes that contributed to plant growth promotion, stress tolerance enhancement, and antimicrobial compound production. B. megaterium HGS7 consistently exhibited antagonistic activity against phytopathogens and strong tolerance to abiotic stress in vitro. Moreover, this strain stimulated mulberry seed germination and seedling growth. It may also induce the production of proline and antioxidant enzymes in mulberry trees to enhance drought tolerance and accelerate growth recovery after drought stress. The knowledge of the interactions between rhizobacteria HGS7 and its host plant might provide a potential strategy to enhance the drought tolerance of mulberry trees in a hydro-fluctuation belt.

Mulberry (Morus alba L.) leaves and fruit are traditional Chinese medicinal materials with anti-inflammatory, immune regulatory, antiviral and anti-diabetic properties. Melatonin performs important roles in the regulation of circadian rhythms and immune activities. We detected, identified and quantitatively analyzed the melatonin contents in leaves and mature fruit from different mulberry varieties. Melatonin and three novel isoforms were found in the Morus plants. Therefore, we conducted an expression analysis of melatonin and its isomer biosynthetic genes and in vitro enzymatic synthesis of melatonin and its isomer to clarify their biosynthetic pathway in mulberry leaves. MaASMT4 and MaASMT20, belonging to class II of the ASMT gene family, were expressed selectively in mulberry leaves, and two recombinant proteins that they expressed catalyzed the conversion of N-acetylserotonin to melatonin and one of three isomers in vitro. Unlike the ASMTs of Arabidopsis and rice, members of the three ASMT gene families in mulberry can catalyze the conversion of N-acetylserotonin to melatonin. This study provides new insights into the molecular mechanisms underlying melatonin and its isomers biosynthesis and expands our knowledge of melatonin isomer biosynthesis.

Abstract Background Miniature inverted-repeat transposable elements (MITEs) are common in eukaryotic genomes, and are important for genomic evolution. Results In the present study, the identification of MITEs in the mulberry genome revealed 286,122 MITE-related sequences, including 90,789 full-length elements. The amplification of mulberry MITEs and the influence of MITEs on the evolution of the mulberry genome were analyzed. The timing of MITE amplifications varied considerably among the various MITE families. Fifty-one MITE families have undergone a single round of amplification, while the other families developed from multiple amplifications. Most mulberry MITEs were inserted near genes and some could regulate gene expression through small RNAs. An analysis of transgenic plants indicated that MITE insertions can upregulate the expression of a target gene. Moreover, MITEs are frequently associated with alternative splicing events (exonizations). Conclusion The data presented herein provide insights into the generation of MITEs as well as their impact on gene regulation and evolution in mulberry.

As a non-essential heavy metal, cadmium (Cd) is toxic to plants. In the last 15 years, over 70 transcriptome studies have been published to decipher the molecular response mechanism against Cd stress in different plants. To extract generalization results from transcriptomic data across different plants and obtain some hub genes that respond to Cd stress, we carried out a meta-analysis of 32 published datasets. Cluster analysis revealed that plant species played a more decisive role than the media used and exposure time in the transcriptome patterns of plant roots response to Cd. The datasets from a Gramineae-like (GL) group were closer in clustering. 838 DEGs were commonly Cd-regulated in at least nine of 18 GL datasets. Gene ontology and KEGG pathway analyses revealed that oxidative stress-related terms and lignin synthesis-related terms were significantly enriched. Mapman analysis revealed that these common DEGs were mainly involved in regulation, cellular response, secondary metabolism, transport, cell wall and lipid metabolism. In Oryza sativa, 15 DEGs were up-regulated in at least four of five HM (As, Cr, Cd, Hg and Pb) groups, such as Os10g0517500 (methionine gamma-lyase) and Os01g0159800 (bHLH107). Moreover, our datasets can be used to retrieve log2FC value of specific genes across 29 studies (48 datasets), which provides data reference for the subsequent selection of HM-related genes. Our results provide the basis for further understanding of Cd tolerance mechanisms in plants.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.

Holometabolous insects have distinct larval, pupal, and adult stages. The pupal stage is typically immobile and can be subject to predation, but cocoon offers pupal protection for many insect species. The cocoon provides a space in which the pupa to adult metamorphosis occurs. It also protects the pupa from weather, predators and parasitoids. Silk protein is a precursor of the silk used in cocoon construction. We used the silkworm as a model species to identify genes affecting silk protein synthesis and cocoon construction. We used quantitative genetic analysis to demonstrate that β-1,4-N-acetylglucosaminidase 1 (BmGlcNase1) is associated with synthesis of sericin, the main composite of cocoon. BmGlcNase1 has an expression pattern coupled with silk gland development and cocoon shell weight (CSW) variation, and CSW is an index of the ability to synthesize silk protein. Up-regulated expression of BmGlcNase1 increased sericin content by 13.9% and 22.5% while down-regulation reduced sericin content by 41.2% and 27.3% in the cocoons of females and males, respectively. Genomic sequencing revealed that sequence variation upstream of the BmGlcNase1 transcriptional start site (TSS) is associated with the expression of BmGlcNase1 and CSW. Selective pressure analysis showed that GlcNase1 was differentially selected in insects with and without cocoons (ω1 = 0.044 vs. ω2 = 0.154). This indicates that this gene has a conserved function in the cocooning process of insects. BmGlcNase1 appears to be involved in sericin synthesis and silkworm cocooning.

The endophytic microbiome is thought to play an important role in promoting plant growth and health. Using culture-independent and culture-dependent protocols, this study characterized the seasonal shifts in the endophytic fungal microbiota of four mulberry (Morus L.) cultivars having different levels of resistance to mulberry fruit sclerotiniosis. Core endophytes can be obtained by two approaches, and they were divided into two clusters by season. Spring samples harbored higher operational taxonomic units (OTUs) and α-diversity, while autumn samples had more sequences or isolates of the fungal class Dothideomycetes with the representative orders Capnodiales and Pleosporales. While comparing different mulberry cultivars, we found that the total number of OTUs in susceptible cultivars was higher than that of resistant cultivars, and Cladosporium sp. were observed in all. Notably, the causal agent of fruit sclerotiniosis (Scleromitrula shiraiana) was only detected in susceptible cultivars. Collectively, our work elucidated significant variations in the mulberry endophytic microbiome, mainly because of seasonal shifts, and the fact that the host cultivars and mulberry endophytic fungal community appeared to have a certain connection with the resistance level of mulberry fruit to sclerotiniosis. These results provided valuable information on the isolation and culturing of mulberry endophytes that could be applied to improve mulberry fruit production and health.

Background Species in the genus Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information. Methods We sequenced the ITS regions and the chloroplast DNA regions TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry. Results We found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades. Conclusions Two species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution.

[This corrects the article DOI: 10.1371/journal.pone.0106962.].

The zinc-regulated transporters, iron-regulated transporter-like proteins (ZIPs), the natural resistance and macrophage proteins (NRAMP), the heavy metal ATPases (HMAs) and the metal tolerance or transporter proteins (MTPs) families are involved in cadmium (Cd) uptake, translocation and sequestration in plants. Mulberry (Morus L.), one of the most ecologically and economically important (as a food plant for silkworm production) genera of perennial trees, exhibits excellent potential for remediating Cd-contaminated soils. However, there is no detailed information about the genes involved in Cd2+ transport in mulberry. In this study, we identified 31 genes based on a genome-wide analysis of the Morus notabilis genome database. According to bioinformatics analysis, the four transporter gene families in Morus were distributed in each group of the phylogenetic tree, and the gene exon/intron structure and protein motif structure were similar among members of the same group. Subcellular localization software predicted that these transporters were mainly distributed in the plasma membrane and the vacuolar membrane, with members of the same group exhibiting similar subcellular locations. Most of the gene promoters contained abiotic stress-related cis-elements. The expression patterns of these genes in different organs were determined, and the patterns identified, allowing the categorization of these genes into four groups. Under low or high-Cd2+ concentrations (30 μM or 100 μM, respectively), the transcriptional regulation of the 31 genes in root, stem and leaf tissues of M. alba seedlings differed with regard to tissue and time of peak expression. Heterologous expression of MaNRAMP1, MaHMA3, MaZIP4, and MaIRT1 in Saccharomyces cerevisiae increased the sensitivity of yeast to Cd, suggested that these transporters had Cd transport activity. Subcellular localization experiment showed that the four transporters were localized to the plasma membrane of yeast and tobacco. These results provide the basis for further understanding of the Cd tolerance mechanism in Morus, which can be exploited in Cd phytoremediation.

Wolbachia naturally infects a wide variety of arthropods, where it plays important roles in host reproduction. It was previously reported that Wolbachia did not infect silkworm. By means of PCR and sequencing we found in this study that Wolbachia is indeed present in silkworm. Phylogenetic analysis indicates that Wolbachia infection in silkworm may have occurred via transfer from parasitic wasps. Furthermore, Southern blotting results suggest a lateral transfer of the wsp gene into the genomes of some wild silkworms. By antibiotic treatments, we found that tetracycline and ciprofloxacin can eliminate Wolbachia in the silkworm and Wolbachia is important to ovary development of silkworm. These results provide clues towards a more comprehensive understanding of the interaction between Wolbachia and silkworm and possibly other lepidopteran insects.

In insects, abdominal appendages, also called prolegs, vary due to adaptive evolution. Mutations on prolegs within species provide insights to better understand the mechanisms underlying appendage development and diversity. In silkworm Bombyx mori, extra-crescents and degenerated abdominal legs (Edl) mutant, belonging to the E pseudoallele group, is a spontaneous mutation that adds crescents and degenerates prolegs on the third abdominal segment (A3). This mutation may be a homeotic transformation of A3 to A2. In this study, the Edl locus was mapped within approximately a 211 Kb region that is 10 Kb upstream of Bmabdominal-A (Bmabd-A). RT-quantitative PCR (RT-qPCR) and Western blot analysis of Bmabd-A expression showed a slight but significant decrease, while the expression of BmUltrabithorax (BmUbx) was up-regulated in the Edl mutant compared to wildtype (Dazao). Moreover, we also found that BmDistal-less (BmDll), which regulated the development of distal proleg structures, was missing at the tips of the A3 prolegs in the Edl mutant compared to BmDll expression in normally developed prolegs in both the wildtype and mutant. Collectively, we identified approximately a 211 Kb region in the Edl locus that regulates BmUbx and Bmabd-A expression and found that changes in BmUbx and Bmabd-A expression may lead to the loss of distal proleg structures in B. mori.

Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while β-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research.

The insect midgut epithelium is generally lined with a uniquechitin and protein structure, the peritrophic membrane (PM),which facilitates food digestion and protects the gut epithelium.We used gel electrophoresis and mass spectrometry toidentify the extracted proteins from the silkworm PM to obtainan in-depth understanding of the biological function of thesilkworm PM components. A total of 305 proteins, with molecularweights ranging from 8.02 kDa to 788.52 kDa and theisoelectric points ranging from 3.39 to 12.91, were successfullyidentified. We also found several major classes of PMproteins, i.e. PM chitin-binding protein, invertebrate intestinalmucin, and chitin deacetylase. The protein profile provides abasis for further study of the physiological events in the PM ofBombyx mori.

The extraction of Ramulus mori polysaccharides (RMPs) was optimized using response surface methodology (RSM). The optimal process conditions, which gave the highest yield of RMPs (6.25%) were 80 °C, 50 min, and a solid⁻liquid ratio of 1:40 (g/mL), with the extraction performed twice. The RMPs contained seven monosaccharides, namely, mannose, rhamnose; glucuronic acid, glucose, xylose, galactose, and arabinose, in a 1.36:2.68:0.46:328.17:1.53:21.80:6.16 molar ratio. The glass transition and melting temperatures of RMPs were 83 and 473 °C, respectively. RMPs were α-polysaccharides and had surfaces that resembled a porous sponge, as observed by scanning electron microscopy. RMPs inhibited the proliferation of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and showed antioxidant activity (assessed by three different methods), although it was generally weaker than that of vitamin C. RMPs showed anti-inflammatory activity in a concentration-dependent manner. This study provides a basis for exploring the potential uses of RMPs.

We analyzed the anti-inflammatory and antinociceptive activities of total flavonoids (TF) found in black mulberry fruits. The TF content was 20.9 mg/g (dry weight). Two anthocyanins, cyanidin-3-O-glucoside (8.3 mg/g) and cyanidin-3-O-rutinoside (2.9 mg/g), were identified in the fruits by UPLC. The TF of black mulberry fruits had significant reducing power and radical (OH(-), O2(·-), DPPH and ABTS) scavenging activities that was demonstrated in a dose-response curve. The TF had inhibitory activities on xylene-induced ear edema and carrageenan-induced paw edema in mice. In addition, TF had antinociceptive activities in the two nociceptive phases of formalin test. We used ELISA to detect the pro-inflammatory cytokines IL-1β, TNF-α, IFN-γ, and NO in the serum of mice. These cytokines were significantly inhibited or scavenged by TF (50 and 100 mg/kg). The results demonstrated that TF of black mulberry possess anti-inflammatory and analgesic effects that might correlate to its antioxidant activities and inhibition of pro-inflammatory cytokines.

Mulberry, belonging to the order Rosales, family Moraceae, and genus Morus, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus Morus has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify Morus species, resulting in varied numbers of designated Morus spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus Morus. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. M. alba and M. notabilis showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the M. notabilis genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the Morus genus should be classified into eight species, including M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus Morus.

Anthocyanins and flavones are important pigments responsible for the coloration of fruits. Mulberry fruit is rich in anthocyanins and flavonols, which have multiple uses in traditional Chinese medicine. The antinociceptive and antibacterial activities of total flavonoids (TF) from black mulberry (MnTF, TF of Morus nigra) and non-black mulberry (MmTF, TF of Morus mongolica; and MazTF, TF of Morus alba ‘Zhenzhubai’) fruits were studied. MnTF was rich in anthocyanins (11.3 mg/g) and flavonols (0.7 mg/g) identified by ultra-performance liquid chromatography–tunable ultraviolet/mass single-quadrupole detection (UPLC–TUV/QDa). Comparatively, MmTF and MazTF had low flavonol contents and MazTF had no anthocyanins. MnTF showed significantly higher antinociceptive and antibacterial activities toward Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus than MmTF and MazTF. MnTF inhibited the expression of interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), phospho-p65 (p-p65) and phospho-IκBα (p-IκBα), and increased interleukin 10 (IL-10). Additionally, mice tests showed that cyanidin-3-O-glucoside (C3G), rutin (Ru) and isoquercetin (IQ) were the main active ingredients in the antinociceptive process. Stronger antinociceptive effect of MnTF was correlated with its high content of anthocyanins and flavonols and its inhibitory effects on proinflammatory cytokines, iNOS and nuclear factor-κB (NF-κB) pathway-related proteins.

Histone methylation plays an important role in gene transcription and chromatin organization and is linked to the silencing of a number of critical tumor suppressor genes in tumorigenesis. G9a is a histone methyltransferase (HMTase) for histone H3 lysine 9. In this study, we investigated the role of G9a in neuroblastoma tumor growth together with the G9a inhibitor BIX01294. The exposure of neuroblastoma cells to BIX01294 resulted in the inhibition of cell growth and proliferation, and BIX01294 treatment resulted in the inhibition of the tumorigenicity of neuroblastoma cells in NOD/SCID mice. Therefore, G9a may be a potential therapeutic target in neuroblastoma. Moreover, we found several specific characteristics of autophagy after BIX01294 treatment, including the appearance of membranous vacuoles and microtubule-associated protein light chain 3 (LC3B). Similar results were observed in G9a-knockdown cells. In conclusion, our results demonstrated that G9a is a prognostic marker in neuroblastoma, and revealed a potential role of G9a in regulating the autophagy signaling pathway in neuroblastoma.

Leflunomide as an immunosuppressive drug is generally used in the treatment of rheumatoid arthritis. It inhibits DHODH (dihydroorotate dehydrogenase ), which is one of the essential enzymes in the de novo pyrimidine biosynthetic pathway. Here we showed that leflunomide significantly reduced cell proliferation and self-renewal activity. Annexin V-FITC/PI staining assay revealed that leflunomide induced S-phase cell cycle arrest, and promoted cell apoptosis. In vivo xenograft study in SCID mice showed that leflunomide inhibited tumor growth and development. We also observed that DHODH was commonly expressed in neuroblastoma. When treated with leflunomide, the neuroblastoma cell lines BE(2)-C, SK-N-DZ, and SK-N-F1 showed dramatic inhibition of DHODH at mRNA and protein levels. Considering the favorable toxicity profile and the successful clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in neuroblastoma.

In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein.

Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild-type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.

Antimicrobial peptides are small-molecule proteins that are usually encoded by multiple-gene families. They play crucial roles in the innate immune response, but reports on the functional divergence of antimicrobial peptide gene families are rare. In this study, 14 paralogs of antimicrobial peptides belonging to cecropin, moricin and gloverin families were recombinantly expressed in pET expression systems. By antimicrobial activity tests, peptides representing paralogs in the same family of cecropin and moricin families, displayed remarkable differences against 10 tested bacteria. The evolutionary rates were relatively fast in the two families, which presented obvious functional divergence among paralogs of each family. Four peptides of gloverin family had similar antimicrobial spectrum and activity against tested bacteria. The gloverin family showed similar antimicrobial function and slow evolutionary rates. By induced transcriptional activity, genes encoding active antimicrobial peptides were upregulated at obviously different levels when silkworm pupae were infected by three types of microbes. Association analysis of antimicrobial activities and induced transcriptional activities indicated that the antimicrobial activities might be positively correlated with induced transcriptional activities in the cecropin and moricin families. These results suggest that representative BmcecB6, BmcecD and Bmmor as the major effector genes have broad antimicrobial spectrum, strong antimicrobial activity and high microbe-induced expression among each family and maybe play crucial roles in eliminating microbial infection.