Andrographolide inhibits the activation of spinal microglia and ameliorates mechanical allodynia.

Andrographolide (Andro), a labdane diterpene, possesses anti-inflammatory properties and has been used to treat numerous inflammatory diseases. Novel findings revealed that Andro might be vital in regulating pain. However, the contribution of Andro to chronic inflammatory pain has yet to be determined, and its underlying mechanism of action remains unknown. In this study, we observed that Andro attenuated mechanical allodynia in inflammatory pain mice induced by injecting complete Freund's adjuvant (CFA) into the right hind paws. This analgesic effect of Andro is mainly dependent on its inhibition of microglial overactivation and the release of proinflammatory cytokines (TNF and IL-1ß) in lumbar spinal cords of inflammatory pain model mice. More importantly, our data in vivo and in vitro revealed a negative role for Andro in regulating the TLR4/NF-κB signaling pathway, which might contribute to the inhibition of spinal microglial activation and proinflammatory cytokines production, and the improvement of paw withdrawal thresholds in a mouse model of chronic inflammatory pain evoked by CFA. We further found the potential interaction of Andro with TLR4/myeloid differentiation factor 2 heterodimer using molecular modeling, implying that TLR4 might be a potential target for Andro to exert an analgesic effect. Taken together, our findings demonstrated that the modulation of spinal microglial activation by Andro might be substantially conducive to managing chronic pain triggered by neuroinflammation.

Gene regulatory network from cranial neural crest cells to osteoblast differentiation and calvarial bone development.

Calvarial bone is one of the most complex sequences of developmental events in embryology, featuring a uniquely transient, pluripotent stem cell-like population known as the cranial neural crest (CNC). The skull is formed through intramembranous ossification with distinct tissue lineages (e.g. neural crest derived frontal bone and mesoderm derived parietal bone). Due to CNC's vast cell fate potential, in response to a series of inductive secreted cues including BMP/TGF-ß, Wnt, FGF, Notch, Hedgehog, Hippo and PDGF signaling, CNC enables generations of a diverse spectrum of differentiated cell types in vivo such as osteoblasts and chondrocytes at the craniofacial level. In recent years, since the studies from a genetic mouse model and single-cell sequencing, new discoveries are uncovered upon CNC patterning, differentiation, and the contribution to the development of cranial bones. In this review, we summarized the differences upon the potential gene regulatory network to regulate CNC derived osteogenic potential in mouse and human, and highlighted specific functions of genetic molecules from multiple signaling pathways and the crosstalk, transcription factors and epigenetic factors in orchestrating CNC commitment and differentiation into osteogenic mesenchyme and bone formation. Disorders in gene regulatory network in CNC patterning indicate highly close relevance to clinical birth defects and diseases, providing valuable transgenic mouse models for subsequent discoveries in delineating the underlying molecular mechanisms. We also emphasized the potential regenerative alternative through scientific discoveries from CNC patterning and genetic molecules in interfering with or alleviating clinical disorders or diseases, which will be beneficial for the molecular targets to be integrated for novel therapeutic strategies in the clinic.

Qualitative Analysis of Visible Foreign Solids in Armillarisin A Injection Formulations Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

During the trial production of Armillarisin A for injection (AA-I), unidentified needle-like yellow-brown crystals were occasionally observed. Here, we report an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method for determining the source of the visible foreign bodies in the formulations of Armillarisin A active pharmaceutical ingredient (AA-API). AA-API, photolyzed samples, the intermediate polymer, and the excipient analyzed determined after the separation on a Waters Symmetry C18 (3.5 µm, 4.6 × 75 mm) column with a mobile phase consisting of a methanol/acetic acid (0.1 mol/L) aqueous solution (50:50). Furthermore, the crystal type of the visible foreign bodies, the intermediate polymer and AA-API were investigated by X-ray powder diffraction (XRD). The results revealed that the characteristics of the visible foreign solids were the same as those of AA-API as regards UPLC peak position (368 nm) and MS spectrum in negative ion detection mode. The visible foreign solids were thus identified as unpolymerized crystals of AA-API and were attributed to AA-API itself. The results showed that the production process could be improved by changing the stirring method and frequency as well as by optimizing the polymerization temperature to ensure the safety, stability, and control of the product quality in the stage of batch production.

Bacillus amyloliquefaciens-9 Reduces Somatic Cell Count and Modifies Fecal Microbiota in Lactating Goats.

Subclinical mastitis is one of the major problems affecting dairy animals' productivity and is classified based on milk somatic cell counts (SCC). Previous data showed that marine-derived Bacillus amyloliquefaciens-9 (GB-9) improved the immunity and the nonspecific immune defense system of the body. In this study, the potential role of GB-9 in improving subclinical mastitis was assessed with Radix Tetrastigmae (RT) as a positive control in subclinical mastitis Saanen dairy goats. The current data showed that GB-9 and RT significantly reduced the SCC in dairy goats. After being fed with GB-9 or RT, the decreased concentrations of malondialdehyde, IgA, IgM, IL-2, IL-4, and IL-6 were observed. The amplicon sequencing analysis of fecal samples revealed that GB-9 significantly altered the bacterial community. Bacteroides and Phascolarctobacterium were the major genera that respond to GB-9 feeding. The correlation analysis using weighted gene co-expression network analysis showed a MePink module was most associated with the serum concentrations of immunoglobulin and interleukin. The MePink module contained 89 OTUs. The feeding of GB-9 in decreasing the SCC was associated with the altered abundance of Bacteroides, which was correlated with the concentrations of immunoglobulins and chemokines. Collectively, the current data suggested that marine-derived GB-9 could be a helpful probiotic to control subclinical mastitis.

Sequence structure character of IgNAR Sec in whitespotted bamboo shark (Chiloscyllium plagiosum).

Whitespotted bamboo shark (Chiloscyllium plagiosum) is a demersal cartilaginous fish with an adaptive immune system founded upon immunoglobulins. In this manuscript, we characterize the IgNAR of the whitespotted bamboo shark. A newly discovered alternative splicing form of IgNAR Sec (IgNARshort (ΔC2-C3) Sec) was identified, in which the C1 domain was spliced directly to the C4 domain, the process resulted in a molecule containing three constant domains. However, a single unpaired cysteine remains in the highly flexible hinge region, contributing in the formation of an interchain disulfide bond. Two types of C1 domain were found, and the one lacking a short α-helix showed lower proportion. This finding suggests that short α-helices might be important to the stability of IgNAR. High-throughput sequencing revealed that the percentage of VNAR types significantly vary between the diverse species of sharks. The variable region of IgNAR (the VNAR) with small size and stabilization is a potential candidate for immunotherapeutic agents. The structure and stability analysis in this manuscript may be useful in future biomedical applications.

Two C-type lectins (ReCTL-1, ReCTL-2) from Rimicaris exoculata display broad nonself recognition spectrum with novel carbohydrate binding specificity.

C-type lectins are Ca2+-dependent carbohydrate-binding proteins containing one or more carbohydrate-recognition domains (CRDs). C-type lectins play crucial roles in innate immunity, including nonself-recognition and pathogen elimination. In the present study, two C-type lectins (designated ReCTL-1 and ReCTL-2) were identified from the shrimp Rimicaris exoculata which dwells in deep-sea hydrothermal vents. The open reading frames of ReCTL-1 and ReCTL-2 encoded polypeptides of 171 and 166 amino acids respectively, which were both composed of a signal peptide and a single CRD. The key motifs determining the carbohydrate binding specificity of ReCTL-1 and ReCTL-2 were respectively Glu-Pro-Ala (EPA) and Gln-Pro-Asn (QPN), which were firstly discovered in R. exoculata. ReCTL-1 and ReCTL-2 displayed similar pathogen-associated molecular pattern (PAMP) binding features and they bound three PAMPs-ß-glucan, lipopolysaccharide and peptidoglycan-with relatively high affinity. In addition, both could efficiently recognize and bind Gram-positive bacteria, Gram-negative bacteria and fungi. However, ReCTL-1 and ReCTL-2 exhibited different microbial agglutination activities: ReCTL-1 agglutinated Staphylococcus aureus and Saccharomyces cerevisiae, while ReCTL-2 agglutinated Micrococcus luteus, Vibrio parahaemolyticus and V. fluvialis. Both ReCTL-1 and ReCTL-2 inhibited the growth of V. fluvialis. All these results illustrated that ReCTL-1 and ReCTL-2 could function as important pattern-recognition receptors with broad nonself-recognition spectra and be involved in immune defense against invaders, but their specificities are not the same. In addition, the two ReCTLs possessed different carbohydrate binding specificities from each other and from the classical pattern: ReCTL-1 with an EPA motif bound d-galactose and l-mannose, while ReCTL-2 with a QPN motif bound d-fucose and N-acetylglucosamine.

Evaluation of the Antibacterial Material Production in the Fermentation of Bacillus amyloliquefaciens-9 from Whitespotted Bamboo Shark (Chiloscyllium plagiosum).

Bacillus amyloliquefaciens-9 (GBacillus-9), which is isolated from the intestinal tract of the white-spotted bamboo shark (Chiloscyllium plagiosum), can secrete potential antibacterial materials, such as ß-1,3-1,4-glucanase and some antimicrobial peptides. However, the low fermentation production has hindered the development of GBacillus-9 as biological additives. In this study, the Plackett-Burman design and response surface methodology were used to optimize the fermentation conditions in a shake flask to obtain a higher yield and antibacterial activity of GBacillus-9. On the basis of the data from medium screening, M9 medium was selected as the basic medium for fermentation. The data from the single-factor experiment showed that sucrose had the highest antibacterial activity among the 10 carbon sources. The Plackett-Burman design identified sucrose, NH4Cl, and MgSO4 as the major variables altering antibacterial activity. The optimal concentrations of these compounds to enhance antibacterial activity were assessed using the central composite design. Data showed that sucrose, NH4Cl, and MgSO4 had the highest antibacterial activities at concentrations of 64.8, 1.84, and 0.08 g L-1, respectively. The data also showed that the optimal fermentation conditions for the antibacterial material production of GBacillus-9 were as follows: Inoculum volume of 5%, initial pH of 7.0, temperature of 36 °C, rotating speed of 180 rpm, and fermentation time of 10 h. The optimal fermentation medium and conditions achieved to improve the yield of antibacterial materials for GBacillus-9 can enhance the process of developing biological additives derived from GBacillus-9.

A fragment activity assay reveals the key residues of TBC1D15 GTPase-activating protein (GAP) in Chiloscyllium plagiosum.

BACKGROUND: GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of our research group in 2017 (Chen et al. in Protein Sci 26(4):834-846, 2017). The protein crystal structure and sequencing data accession numbers in Protein structure database (PDB) are 5TUB (Shark TBC1D15 GAP) and 5TUC (Sus TBC1D15 GAP), respectively. In this paper, we analyzed the Rab-GAP specificity of TBC1D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity. RESULTS: Sequence alignment showed that five arginine residues of the TBC1D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark. A fragment activity assay was conducted by altering the five residues of Shark TBC1D15-GAP to arginine, and the corresponding arginine in TBC1D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1D15-GAP. Our data revealed that the residues of G28, K45, K119, K122 and K221 in the Shark TBC1D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11. Mutation of the five residues significantly altered the Shark TBC1D15-GAP activity. CONCLUSIONS: These results revealed that the substrate specificity of TBC1D15 has had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals. Collectively, the data support a different mechanism of Shark TBC1D15-GAP in substrate selection, which provides a new idea for the development of Marine drugs.

Dulaglutide inhibits high glucose- induced endothelial dysfunction and NLRP3 inflammasome activation.

Activation of the NLRP3 inflammasome plays an important role in high glucose- induced endothelial dysfunction in patients with type 2 diabetes mellitus (T2DM). Dulaglutide, a newly developed glucagon-like peptide-1 receptor (GLP-1R) agonist, has been approved for the management of T2DM. In the current study, we aimed to investigate whether dulaglutide possesses a protective effect against high glucose- induced activation of the NLRP3 inflammasome. Our results indicate that dulaglutide treatment prevented high glucose- induced generation of reactive oxygen species (ROS) and protein carbonyl, as well as the expression of NADPH oxidase 4 (NOX-4) in human umbilical vein endothelial cells (HUVECs). Dulaglutide treatment could inhibit high glucose- induced release of lactate dehydrogenase (LDH) and the expression of TXNIP. Dulaglutide suppressed high glucose- induced activation of NLRP3 inflammasome by reducing the expression of NLRP3, ASC, and cleaved caspase 1 (P10). Notably, dulaglutide treatment suppressed high glucose- induced maturation of IL-1ß and IL-18. Mechanistically, our findings indicate that SIRT1 was involved in this process by showing that knockdown of SIRT1 by transfection with SIRT1 siRNA abolished the inhibitory effects of dulaglutide on IL-1ß and IL-18 secretion via suppression of NLRP3, ASC, and p10. These data suggest that dulaglutide might serve as a potential drug for the treatment of cardiovascular complications in T2DM patients.

cGAMP inhibits tumor growth in colorectal cancer metastasis through the STING/STAT3 axis in a zebrafish xenograft model.

The leading cause of mortality due to colorectal cancer (CRC) is highly associated with the development of liver metastases. Recently, we described cGAMP that is closely related to the metastatic state wherein the progress of metastatic tumors is associated with favorable outcomes in a zebrafish xenograft model. cGAMP was administered and the expression levels of type-I interferons were induced amongst tumor tissues to illuminate the overall measure of the induced STING/STAT3 axis in colorectal liver metastases. Furthermore, cGAMP-STING dependent STAT3 activation resulted in the inhibition of tumor cell proliferation, viability, and invasion in vitro. The subtotal reduction in tumor growth attributed to a large number of infiltrating inflammatory cells in vivo. We showed that cGAMP inhibited migration through angiogenesis by up-regulating IL-2, TNF-α, and IFN-γ, whereas STAT3 down-regulation inhibited CXCL8, BCL-2, and VEGFA expression. The importance of cGAMP in inhibiting the invasion front of CRC confirmed that the cGAMP dependent activation of STING/STAT3 axis played a key role in the inhibition of tumor progression.

Characterisation and functional comparison of single-CRD and multidomain containing galectins CgGal-2 and CgGal-3 from oyster Crassostrea gigas.

Galectins are ß-galactoside binding lectins that play crucial roles in innate immunity in vertebrates and invertebrates through their conserved carbohydrate-recognition domains (CRDs). In the present study, single- and four-CRD-containing galectins were identified in oyster Crassostrea gigas (designated CgGal-2 and CgGal-3). The open reading frames (ORFs) of CgGal-2 and CgGal-3 encode polypeptides of 200 and 555 amino acids, respectively. All CRDs of CgGal-3 include two consensus motifs essential for ligand-binding, and a novel motif is present in CgGal-2. Pathogen-associated molecular pattern (PAMP) profiles were determined for recombinant rCgGal-2 and rCgGal-3, and rCgGal-2 displayed low binding affinity for PAMPs, while rCgGal-3 bound various PAMPs including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN) with relatively high affinity. Furthermore, rCgGal-2 and rCgGal-3 exhibited different microbe binding profiles; rCgGal-2 bound to Gram-negative bacteria (Escherichia coli and Vibrio vulnificus) and fungi (Saccharomyces cerevisiae and Pichia pastoris), while rCgGal-3 bound to these microbes but also to Gram-positive bacteria (Micrococcus luteus). In addition, rCgGal-3 possessed microbial agglutinating activity and coagulation activity against fungi and erythrocytes, respectively, but rCgGal-2 lacked any agglutinating activity. Carbohydrate binding specificity analysis showed that rCgGal-3 specifically bound D-galactose. Furthermore, rCgGal-2 and rCgGal-3 functioned as opsonin participating in the clearance against invaders in C. gigas. Thus, CgGal-2 with one CRD and CgGal-3 with four CRDs are new members of the galectin family involved in immune responses against bacterial infection. Differences in the organisation and amino acid sequences of CRDs may affect their specificity and affinity for nonself substances.

Identification of Selective, Cell Active Inhibitors of Protein Arginine Methyltransferase 5 through Structure-Based Virtual Screening and Biological Assays.

Protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme, is reported as an important therapeutic target in leukemia and lymphoma. In the present study, based on the combination of virtual screening and biochemical validations, we discovered a series of small-molecule inhibitors targeting PRMT5. Among those, DC_Y134 exhibited the most potent activity with IC50 value of 1.7 µM and displayed good selectivity against other methyltransferases. Further treatment with DC_Y134 inhibited the proliferation of several hematological malignancy cell lines by causing cell cycle arrest and apoptosis. Western blot assays indicated that DC_Y134 reduced the cellular symmetrically dimethylated levels. In addition, we analyzed the binding mode of DC_Y134 through molecular docking, which revealed that DC_Y134 occupies the binding site of substrate arginine and explained the selectivity of this inhibitor. Taken together, compound DC_Y134 could be used to elucidate the biological roles of PRMT5 and serve as a lead compound for treatment of hematologic malignancies.

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.

Hyphococcus flavus gen. nov., sp. nov., a novel alphaproteobacterium isolated from deep seawater.

A Gram-staining-negative, aerobic, non-spore-forming, coccoid to rod shaped bacteria with prosthecate and flagellum, designated as HSF6T, was isolated from deep seawater samples collected from the South China Sea at depth of 2.5 km and subjected to a polyphasic taxonomic investigation. Colonies of strain HSF6T were 1-2 mm in diameter, smooth, circular, convex and yellow. Strain HSF6T was found to grow at 15-37 °C (optimum, 25-35 °C), pH 5.0-9.5 (optimum, pH 7.0-7.5) and with 0-8 % (w/v) NaCl (optimum, 2 %). Chemotaxonomic analysis showed the predominant respiratory quinone of strains HSF6T were ubiquinone-10, and the major fatty acids were C18 : 1ω7c, C16 : 0 and 11-methyl C18 : 1ω7c. The polar lipids were monoglycosyldiglyceride (MGDG), sulfo-quinovosyl diacylglycerol (SQDG), three unknown glycolipids (GL1-3) and five unknown lipids (L1-5). The DNA G+C content of strain HSF6T was determined to be 51.0 mol% with HPLC. The comparison of 16S rRNA gene sequence similarities show that strain HSF6T was related most closely to genus Parvularcula with similarity ranging from 91.0 to 91.8 %. The phylogenetic trees, using the 16S rRNA gene sequence, reconstructed with neighbour-joining, maximum-parsimony and maximum-likelihood methods showed that strain HSF6T constituted a separated branch in the family 'Parvularculaceae'. Differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain HSF6T is clearly distinct from validly published genera. On the basis of these features, we propose strain HSF6T (=MCCC 1K03223T=KCTC 52486T) represents a novel species of a novel genus with the name Hyphococcus flavus gen. nov., sp. nov.

Hyphobacterium vulgare gen. nov., sp. nov., a novel alphaproteobacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-spore-forming, non-motile, oval to rod-shaped, prosthecate bacterium, designated strain WM6T, was isolated from a seawater sample collected from the South China Sea at a depth of 150 m and subjected to a polyphasic taxonomic investigation. Cells of strain WM6T were approximately 0.5-0.6 µm in width and 0.8-1.2 µm in length, and colonies were smooth, circular, convex and whitish yellow. Strain WM6T was found to grow at 10-45 °C (optimum, 30 °C), at pH 6.5-9.0 (optimum, pH 7.5-8.5) and with 1-6 % (w/v) NaCl (optimum, 1-2 %). Chemotaxonomic analysis showed the predominant respiratory quinone and the major fatty acid of strains WM6T were ubiquinone-10 and C18 : 1ω7c, respectively. The polar lipids of strain WM6T were phosphatidylglycerol, glucuronopyranosyldiglyceride, monoglycosyldiglyceride, sulfo-quinovosyl diacylglycerol, seven unknown glycolipids and two unknown lipids. The DNA G+C content of strain WM6T was determined to be 59.8 mol% by HPLC. 16S rRNA gene sequence similarities showed that strain WM6T was related most closely to the genus Maricaulis with a similarity range from 92.3 to 93.8 %. Phylogenetic trees reconstructed with the neighbour-joining and maximum-likelihood methods using mega and maximum-likelihood methods using arb showed that strain WM6T constituted a separated branch in the family Hyphomonadaceae. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain WM6T is clearly distinct from any validly published genus. On the basis of these features, strain WM6T represents a novel species of a new genus with the name Hyphobacterium vulgare gen. nov., sp. nov. The type strain of Hyphobacterium vulgare is WM6T (=MCCC 1K03222T=KCTC 52487T).

The functional characterization and comparison of two single CRD containing C-type lectins with novel and typical key motifs from Portunus trituberculatus.

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads.

Differential expression and miRNA regulation of the GSTP1 gene in the regenerating liver of Chiloscyllium plagiosum.

Liver regeneration is a complicated process, and understanding the regulatory mechanism will be helpful in the treatment of diseases associated with liver. In this study, the one-third liver resection model was established in Chiloscyllium plagiosum, and the whole transcriptome of the C. plagiosum was generated using the Illumina-Solexa sequencing platform. Differentially expressed genes were analyzed using bioinformatics methods and verified using quantitative real-time PCR (qRT-PCR). Using miRanda and TargetScan, we screened the microRNA library for miRNAs that target the glutathione S-transferase P1(GSTP1) gene. Dual-luciferase reporter assays were used to confirm binding between the miRNA and GSTP1. Finally, we used western blotting analysis to determine expression of the GSTP1 protein. As a result, 65,356 unigenes were obtained in normal and damaged liver tissues, with mean length of 955 bp. A total of 359 differentially expressed genes were acquired; 217 of which were upregulated, and 142 were downregulated, including the GSTP1 gene, following liver resection. The presence of the GSTP1 protein in C. plagiosum was shown for the first time. Luciferase reporter assay revealed that GSTP1 messenger RNA was targeted by ipu-miR-143. The discovery and differential expression analysis of GSTP1 in C. plagiosum will be a valuable resource to explain the molecular mechanism of GSTP1 regulation of liver repair.

BIOINFORMATICS, TISSUE DISTRIBUTION, AND SUBCELLULAR LOCALIZATION ANALYSES OF FK506 BINDING PROTEIN 12B FROM SILKWORMS.

FK506 binding proteins (FKBPs) are intracellular receptors of the immunosuppressant FK506 and play important roles in the correct folding of new proteins and the self-assembly of biological macromolecules. FKBP12 is a member of the FKBP family that is widely expressed and highly conserved in many species. In this study, we identified the complete cDNA sequence encoding the FKBP12 ortholog in Bombyx mori, named Bm-FKBP12B (GenBank accession no. DQ443423). Multiple-sequence alignment among different species revealed a high similarity among FKBP12 paralogs and orthologs. Bioinformatics analysis of the Bm-FKBP12B gene showed that it is located on chromosome 20 and consists of three exons and two introns. We cloned, expressed, and purified the Bm-FKBP12B protein in Escherichia coli and generated a specific polyclonal antibody against Bm-FKBP12B. The real-time quantitative reverse-transcription (qRT) PCR and Western blotting results showed that Bm-FKBP12B was present throughout all of the development stages, but it was abundant in the adult and embryo stages. Bm-FKBP12B expression was higher in the silk gland and gut, suggesting that it might play important roles in regulating gene expression in the silk gland and during silk fiber formation. Bm-FKBP12B protein was distributed in the cytoplasm, nucleus, and nuclear membrane.

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.