The fall armyworm (FAW), Spodoptera frugiperda, is a destructive pest native to America and has recently become an invasive insect pest in China. Because of its rapid spread and great risks in China, understanding of FAW genetic background and pesticide resistance is urgent and essential to develop effective management strategies. Here, we assembled a chromosome-level genome of a male FAW (SFynMstLFR) and compared re-sequencing results of the populations from America, Africa, and China. Strain identification of 163 individuals collected from America, Africa and China showed that both C and R strains were found in the American populations, while only C strain was found in the Chinese and African populations. Moreover, population genomics analysis showed that populations from Africa and China have close relationship with significantly genetic differentiation from American populations. Taken together, FAWs invaded into China were most likely originated from Africa. Comparative genomics analysis displayed that the cytochrome p450 gene family is extremely expanded to 425 members in FAW, of which 283 genes are specific to FAW. Treatments of Chinese populations with twenty-three pesticides showed the variant patterns of transcriptome profiles, and several detoxification genes such as AOX, UGT and GST specially responded to the pesticides. These findings will be useful in developing effective strategies for management of FAW in China and other invaded areas.
Animals ; China ; Genomics ; Humans ; Male ; Pesticides ; Spodoptera/genetics* ; Transcriptome

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
Animals ; Baculoviridae ; Genetic Vectors ; Humans ; Recombinant Proteins ; Sf9 Cells ; Spodoptera

ATP-sensitive potassium channels (K) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.
Adenosine Triphosphate ; metabolism ; Amino Acid Sequence ; Animals ; Binding Sites ; Cryoelectron Microscopy ; Ligands ; Mesocricetus ; Mice ; Models, Molecular ; Nucleotides ; metabolism ; Pancreas ; metabolism ; Potassium Channels, Inwardly Rectifying ; chemistry ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Subunits ; chemistry ; metabolism ; Sf9 Cells ; Spodoptera ; Sulfonylurea Receptors ; chemistry ; metabolism

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.
Animals ; Computational Biology ; Crystallography, X-Ray ; Gene Expression ; Humans ; Plasmids ; genetics ; metabolism ; Protein Domains ; Receptors, Adrenergic, beta-1 ; Receptors, G-Protein-Coupled ; classification ; genetics ; metabolism ; Receptors, Odorant ; metabolism ; Receptors, Purinergic P1 ; genetics ; metabolism ; Sf9 Cells ; Spodoptera

The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.
Amino Acid Sequence ; Animals ; Baculoviridae ; genetics ; Codon ; Gene Expression Profiling ; Gene Expression Regulation ; Glycosylation ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sf9 Cells ; Spodoptera ; genetics

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.
Avulavirus* ; Baculoviridae* ; Blotting, Western ; Clone Cells ; Cross Reactions ; Diagnosis ; Ducks ; Glycosylation ; Hemagglutination ; HN Protein ; Immune Sera ; Insects ; N-Acetylneuraminic Acid ; Serologic Tests ; Serotyping ; Spodoptera

In the search for novel potent fungi-derived bioactive compounds for bioinsecticide applications, crude ethyl acetate culture filtrate extracts from 110 mangrove fungal endophytes were screened for their toxicity. Toxicity tests of all extracts against brine shrimp (Artemia salina) larvae were performed. The extracts with the highest toxicity were further examined for insecticidal activity against Spodoptera litura larvae and acetylcholinesterase (AChE) inhibition activity. The results showed that the extracts of five isolates exhibited the highest toxicity to brine shrimp at 50% lethal concentration (LC50) values of 7.45 to 10.24 ppm. These five fungal isolates that obtained from Rhizophora mucronata were identified based on sequence data analysis of the internal transcribed spacer region of rDNA as Aspergillus oryzae (strain BPPTCC 6036), Emericella nidulans (strains BPPTCC 6035 and BPPTCC 6038), A. tamarii (strain BPPTCC 6037), and A. versicolor (strain BPPTCC 6039). The mean percentage of S. litura larval mortality following topical application of the five extracts ranged from 16.7% to 43.3%. In the AChE inhibition assay, the inhibition rates of the five extracts ranged from 40.7% to 48.9%, while eserine (positive control) had an inhibition rate of 96.8%, at a concentration of 100 ppm. The extracts used were crude extracts, so their potential as sources of AChE inhibition compounds makes them likely candidates as neurotoxins. The high-performance liquid chromatography profiles of the five extracts differed, indicating variations in their chemical constituents. This study highlights the potential of culture filtrate ethyl acetate extracts of mangrove fungal endophytes as a source of new potential bioactive compounds for bioinsecticide applications.
Acetylcholinesterase ; Artemia ; Aspergillus oryzae ; Chromatography, Liquid ; Complex Mixtures ; DNA, Ribosomal ; Emericella ; Endophytes* ; Larva ; Mortality ; Neurotoxins ; Physostigmine ; Rhizophoraceae ; Spodoptera ; Statistics as Topic ; Toxicity Tests

PURPOSE: Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Recent genotype (G) shift phenomenon (G3 to G1) in the Asia-wide has posed a challenge for proper prevention by the current vaccine strain. Thus, new kinds of JEV G1 vaccines with enhanced immunogenicity have been required for pigs. MATERIALS AND METHODS: Recombinant porcine granulocyte monocyte-colony stimulating factor (reporGM-CSF) protein was expressed in Spodoptera frugiperda (Sf-9) cells using baculovirus expression system. Two kinds of trials with inactivated JEV vaccines containing IMS1313 adjuvant (Seppic, France) were prepared with or without reporGM-CSF protein. Safety and immunogenicity of the pigs inoculated with the JEV vaccines via intramuscular route was evaluated for 28 days after inoculation. RESULTS: Mice, guinea pigs, and fattening pigs inoculated with the inactivated vaccine showed no signs for 14 and 21 days. Both hemagglutination inhibition and plaque reduction neutralizing antibody titers were significantly higher in pigs immunized with the vaccine containing reporGM-CSF protein after boosting. However, on the side of vaccine efficacy, most mice (87%) immunized with the inactivated JEV vaccine survived after virulent JEV challenge. Whereas the group with the vaccine containing reporGM-CSF protein showed lower protective effects than the vaccine alone for the biological activity of the GM-CSF depending on species specific. CONCLUSION: Our data indicate that animals inoculated with the JEV vaccines was safe and pigs inoculated with inactivated JEV vaccine containing reporGM-CSF protein showed higher humoral immune responses than that of inactivated JEV vaccine without reporGM-CSF protein.
Animals ; Antibodies, Neutralizing ; Baculoviridae ; Encephalitis Virus, Japanese ; Encephalitis, Japanese* ; Genotype* ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocytes ; Guinea Pigs ; Hemagglutination ; Immunity, Humoral ; Mice ; Spodoptera ; Swine* ; Vaccines

Protein kinase D (PKD) is a novel family of serine/threonine kinases and diacylglycerol (DAG) receptors and has been documented in a variety of cellular processes. To get high purity catalytic domain of PKD1 (PKD1-cat) for crystallography study, the GST-tagged PKD1-cat gene was cloned into a baculovirus transfer vector pFastBac1 (donor plasmid). When the recombinant plasmid was transformed into DH10Bac competent Escherichia coli, which contains a baculovirus shuttle vector (bacmid), transposition occurs to generate a recombinant bacmid containing the gene of interest (GST-PKD1-cat). The recombinant bacmid DNA was transfected into Spodoptera frugiperda Sf9 insect cells to generate recombinant baculovirus, which was then amplified through multiple rounds of infection in Sf9 cells. After that, Trichoplusia ni insect cells in suspension culture were infected with baculoviral stock at a multiplicity of infection (MOI) of 5 PFU/cell. SDS-PAGE and Western blotting analysis confirmed the detection of a 68 kDa protein by the glutathione S-transferase (GST) monoclonal antibody. The recombination protein was purified by Glutathione sepharose affinity chromatography and cleaved by PreScission Protease to remove GST tag, and a highly pure 42 kDa protein which was consistent with the molecular weight of the expected PKD1-cat protein was detected on SDS-PAGE. The activity of purified PKD1-cat protein was determined by in vitro PKD kinase assay. Our data showed that the kinase activity increased with the concentration of purified PKD1-cat protein. These results showed that the truncated recombinant PKD1-cat protein was highly active and pure, and could potentially be used for solving 3D structure of this protein by Nuclear Magnetic Resonance (NMR) or crystallography.
Animals ; Baculoviridae ; Blotting, Western ; Catalytic Domain ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; Plasmids ; Protein Kinase C ; chemistry ; Recombinant Proteins ; chemistry ; Spodoptera