Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.

Molecular dynamics simulation, ab initio calculation, and size-selected anion photoelectron spectroscopy study of initial hydration processes of calcium chloride.

To understand the initial hydration processes of CaCl2, we performed molecular simulations employing the force field based on the theory of electronic continuum correction with rescaling. Integrated tempering sampling molecular dynamics were combined with ab initio calculations to overcome the sampling challenge in cluster structure search and refinement. The calculated vertical detachment energies of CaCl2(H2O)n- (n = 0-8) were compared with the values obtained from photoelectron spectra, and consistency was found between the experiment and computation. Separation of the Cl-Ca ion pair is investigated in CaCl2(H2O)n- anions, where the first Ca-Cl ionic bond required 4 water molecules, and both Ca-Cl bonds are broken when the number of water molecules is larger than 7. For neutral CaCl2(H2O)n clusters, breaking of the first Ca-Cl bond starts at n = 5, and 8 water molecules are not enough to separate the two ion pairs. Comparing with the observations on magnesium chloride, it shows that separating one ion pair in CaCl2(H2O)n requires fewer water molecules than those for MgCl2(H2O)n. Coincidentally, the solubility of calcium chloride is higher than that of magnesium chloride in bulk solutions.

Initial hydration processes of magnesium chloride: size-selected anion photoelectron spectroscopy and ab initio calculations.

To understand the initial hydration processes of MgCl2, we measured photoelectron spectra of MgCl2(H2O)n- (n = 0-6) and conducted ab initio calculations on MgCl2(H2O)n- and their neutral counterparts up to n = 7. A dramatic drop in the vertical detachment energy (VDE) was observed upon addition of the first water molecule to bare MgCl2-. This large variation in VDE can be associated with the charge-transfer-to-solvent (CTTS) effect occurring in the MgCl2(H2O)n- clusters, as hydration induces transfer of the excess electron of MgCl2- to the water molecules. Investigation of the separation of Cl--Mg2+ ion pair shows that, in MgCl2(H2O)n- anions, breaking of the first Mg-Cl bond occurs at n = 4, while breaking of the second Mg-Cl bond takes place at n = 6. For neutral MgCl2(H2O)n clusters, breaking of the first Mg-Cl bond starts at n = 7.

Mucin-like protein, a saliva component involved in brown planthopper virulence and host adaptation.

The rice brown planthopper (BPH), Nilaparvata lugens, can rapidly adapt to new resistant rice varieties within several generations, rendering its management burdensome. However, the molecular mechanism underlying its adaptability remains unclear. In this study, we investigated the potential role of mucin-like protein (NlMul) in N. lugens virulence and adaptation to host resistance. NlMul is an important glycoprotein that constitutes both gelling and watery saliva, and specifically expressed in the salivary glands at all developmental stages except the egg period. Knocking down the expression of NlMul resulted in the secretion of short and single-branched salivary sheaths. NlMul might help BPH deal with plant resistance, and altered gene expression was observed when BPHs were transferred from a susceptible rice variety to a resistant one. The NlMul-deficient BPHs showed disordered developmental duration and a portion of these insects reared on resistant rice exhibited lethal effects. Our results uncover a saliva-mediated interaction between insect and host plant, and provide useful information in rice breeding and planthopper management.

A mitochondrial membrane protein is a target for rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV; Reoviridae) is exclusively transmitted by the brown planthopper Nilaparvata lugens in a persistent-propagative manner. It is understood that RNA viral proliferation is associated with the intracellular membranes of the insect host cells. However, the molecular mechanisms of the interaction between the RRSV proliferation and the intracellular membranes remain essentially unknown. It will be of great interest to determine whether RRSV protein(s) directly interact with intracellular membrane components of its host cells. In this study, we identified a RRSV nonstructural protein Pns10 interacting with a host oligomycin-sensitivity conferral protein (OSCP) using yeast two-hybrid system. The interaction between RRSV Pns10 and N. lugens OSCP was verified by a glutathione S-transferase pull-down assay. Confocal miscopy revealed colocalization of these two proteins in the cytoplasm of the salivary gland cells during the viral infection. The virions were further detected in the mitochondria under confocal miscopy and transmission electron microscopy combined with western blotting assay. This is the first observation that RRSV protein has a direct link with mitochondria. Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects. These findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane and provide insights concerning the mitochondrial membrane protein-based virus proliferation mode in the insect vector.

Emergence of Solvent-Separated Na+-Cl- Ion Pair in Salt Water: Photoelectron Spectroscopy and Theoretical Calculations.

Solvation of salts in water is a fundamental physical chemical process, but the underlying mechanism remains unclear. We investigated the contact ion pair (CIP) to solvent-separated ion pair (SSIP) transition in NaCl(H2O)n clusters with anion photoelectron spectroscopy and ab initio calculations. It is found that the SSIP type of structures show up at n = 2 for NaCl-(H2O)n anions. For neutral NaCl(H2O)n, the CIP structures are dominant at n < 9. At n = 9-12, the CIP structures and SSIP structures of NaCl(H2O)n are nearly degenerate in energy, coincident to the H2O:NaCl molar ratio of NaCl saturated solution and implying that the CIP and SSIP structures can coexist in concentrated solutions. These results are useful for understanding the solvation of salts at the molecular level.

Screening and Functional Analyses of Nilaparvata lugens Salivary Proteome.

Most phloem-feeding insects secrete gelling and watery saliva during the feeding process. However, the functions of salivary proteins are poorly understood. In this study, our purpose was to reveal the components and functions of saliva in a rice sap-sucking insect pest, Nilaparvata lugens. The accomplishment of the whole genome and transcriptome sequencing in N. lugens would be helpful for elucidating the gene information and expression specificity of the salivary proteins. In this study, we have, for the first time, identified the abundant protein components from gelling and watery saliva in a monophagous sap-sucking insect species through shotgun proteomic detection combined with the genomic and transcriptomic analysis. Eight unknown secreted proteins were limited to N. lugens, indicating species-specific saliva components. A group of annexin-like proteins first identified in the secreted saliva displayed different domain structure and expression specificity with typical insect annexins. Nineteen genes encoding five annexin-like proteins, six salivaps (salivary glands-specific proteins with unknown function), seven putative enzymes, and a mucin-like protein showed salivary gland-specific expression pattern, suggesting their importance in the physiological mechanisms of salivary gland and saliva in this insect species. RNA interference revealed that salivap-3 is a key protein factor in forming the salivary sheath, while annexin-like5 and carbonic anhydrase are indispensable for N. lugens survival. These novel findings will greatly help to clarify the detailed functions of salivary proteins in the physiological process of N. lugens and elucidate the interaction mechanisms between N. lugens and the rice plant, which could provide important targets for the future management of rice pests.

Initial hydration behavior of sodium iodide dimer: photoelectron spectroscopy and ab initio calculations.

We investigated (NaI)2(-)(H2O)n (n = 0-6) clusters to examine the initial solvation process of (NaI)2 in water, using negative ion photoelectron spectroscopy and theoretical calculations. The structures of these clusters and their neutrals were determined by comparing ab initio calculations with experimental results. It is found that bare (NaI)2(-) is a L-shaped structure and the corresponding neutral is a rhombus. In (NaI)2(-)(H2O), the water molecule prefers to interact with the middle Na atom of the L-shaped (NaI)2(-). For (NaI)2(-)(H2O)n clusters with n = 2-3, two types of structures are nearly degenerate in energy: one is L-shaped and the other is pyramid-shaped. As for (NaI)2(-)(H2O)n with n = 4-6, the dominant structures are pyramid-shaped. For the anionic clusters, one of the Na-I distances increases abruptly when n = 2; for the neutral clusters, rapid lengthening of the Na-I distances occurs when n = 4. Additionally, analyses of the reduced density gradient were carried out, and the results reveal that Na(+)-water interactions dominate in (NaI)2(-)(H2O)n for n≤ 4, whereas I(-)-water and water-water interactions are significantly enhanced when n increases to 5.

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants.

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.

Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species.

From thermodynamics to kinetics: enhanced sampling of rare events.

Despite great advances in molecular dynamics simulations, there remain large gaps between the simulations and experimental observations in terms of the time and length scales that can be approached. Developing fast and accurate algorithms and methods is of ultimate importance to bridge these gaps. In this Account, we briefly summarize recent efforts in such directions. In particular, we focus on integrated tempering sampling. The efficiency of this sampling method has been demonstrated by applications to a range of chemical and biological problems: protein folding, molecular cluster structure searches, and chemical reactions. The combination of integrated tempering sampling and a trajectory sampling method allows the calculation of rate constants and reaction pathways without predefined collective coordinates.

Photoelectron spectroscopy and ab initio calculations of Li(H2O)n(-) and Cs(H2O)n(-) (n = 1-6) clusters.

The Li(H2O)n(-) and Cs(H2O)n(-) (n = 0-6) clusters were studied using anion photoelectron spectroscopy combined with ab initio calculations. It was found that Li tends to be surrounded by water molecules with no water-water H-bonds being formed in the first hydration shell; while Cs sticks on the surface of water-water H-bonds network. The Li atom in its anionic or neutral state is surrounded by four water molecules through Li-O interactions within the first hydration shell; while the case of Cs is different. For the anionic Cs(H2O)n(-) clusters, two types of structures, namely H-end and O-end structures, were identified, with nearly degenerate energies. For the neutral Cs(H2O)n clusters, only O-end structures exist and the first hydration shell of the Cs atom has four water molecules. The different hydration nature of Li and Cs atoms can be ascribed to the delicate balance between the alkali metal-water interactions and the water-water interactions as well as the effect of excess electron.

Stable salt-water cluster structures reflect the delicate competition between ion-water and water-water interactions.

How salts affect water structure is an important topic in many research fields. Salt-water clusters can be used as model systems to extract interaction information that is difficult to obtain directly from bulk solutions. In the present study, integrated tempering sampling molecular dynamics (MD) are combined with quantum mechanics (QM) calculations to overcome the sampling problem in cluster structure searches. We used LiI(H2O)n and CsI(H2O)n as representatives to investigate the microsolvation of ion pairs. It was found that Li(+)-I(-) and Cs(+)-I(-) ion pairs interact with water molecules in very different ways, and the corresponding salt-water clusters have distinctly different structures. LiI strongly affects water-water interactions, and the LiI(H2O)n (n ≥ 5) clusters build around a Li(+)(H2O)4 motif. CsI only slightly perturbs the water cluster structure, and CsI(H2O)n favors the clathrate-like structure when n = 18 or 20. Consistent with the law of "matching water affinities", Li(+) and I(-) are more easily separated by solvent molecules than the Cs(+)-I(-) ion pair.

Microsolvation of LiI and CsI in water: anion photoelectron spectroscopy and ab initio calculations.

In order to understand the microsolvation of LiI and CsI in water and provide information about the dependence of solvation processes on different ions, we investigated the LiI(H2O)n(-) and CsI(H2O)n(-) (n = 0-6) clusters using photoelectron spectroscopy. The structures of these clusters and their corresponding neutrals were investigated with ab initio calculations and confirmed by comparing with the photoelectron spectroscopy experiments. Our studies show that the structural evolutions of LiI(H2O)n and CsI(H2O)n clusters are very different. The Li-I distance in LiI(H2O)n(-) increases abruptly at n = 3, whereas the abrupt elongation of the Li-I distance in neutral LiI(H2O)n occurs at n = 5. In contrast to the LiI(H2O)n(-) clusters, the Cs-I distance in CsI(H2O)n(-) increases significantly at n = 3, reaches a maximum at n = 4, and decreases again as n increases further. There is no abrupt change of the Cs-I distance in neutral CsI(H2O)n as n increases from 0 to 6. Water molecules interact strongly with the Li ion; consequently, water molecule(s) can insert within the Li(+)-I(-) ion pair. In contrast, five or six water molecules are not enough to induce obvious separation of the Cs(+)-I(-) ion pair since the Cs-water interaction is relatively weak compared to the Li-water interaction. Our work has shown that the structural variation and microsolvation in MI(H2O)n clusters are determined by the delicate balance between ion-ion, ion-water, and water-water interactions, which may have significant implications for the general understanding of salt effects in water solution.

Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays.

The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.

Induced endothelial cells enhance osteogenesis and vascularization of mesenchymal stem cells.

Adequate vascularization remains one of the major challenges in bone tissue engineering. Since the microvascular endothelium is of benefit to osteogenesis and vascularization when in direct contact with bone marrow mesenchymal stem cells (BM-MSCs), we investigated whether endothelial cells induced from BM-MSCs have the same effect on BM-MSCs in vitro and in vivo. BM-MSCs were isolated, characterized and induced into endothelial-like cells (induced endothelial cells, IECs) in endothelial cell growth medium 2. BM-MSCs and IECs were co-cultured with direct contact. In vitro, IECs were evaluated in terms of their characteristics of endothelial cells and their effects on the osteogenic potential of BM-MSCs by cell morphology, immunofluorescent staining, alkaline phosphatase activity and osteocalcin synthesis. In vivo, scaffolds consisting of beta-tricalcium phosphate co-seeded with IECs and BM-MSCs were transplanted into mouse dorsal pockets, and a histological analysis was performed to determine the extent of new bone and blood vessel formation. Isolated BM-MSCs were positive for the markers CD105 and CD29 and negative for hematopoietic markers CD34, CD45 and CD14. They were able to differentiate into adipocytes, osteocytes and chondrocytes in respective media. Immunofluorescent analysis with von Willebrand factor and CD31 staining showed that BM-MSCs could differentiate into endothelial cells. The alkaline phosphatase activity and the osteocalcin content of the co-culture group were obviously higher than those of any other group (p < 0.05). Histologically, newly formed bone and vessels were more evident in the culture group (p < 0.05). Our findings suggest that IECs could efficiently stimulate the in vitro differentiation of osteoblast-like cells and promote osteogenesis in vivo by direct contact with BM-MSCs.

[Prokaryotic expression, purification and refolding of extracellular ligand binding domains of chick Tie-2 and its immunogenicity].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick Tie-2, the purification and refolding conditions of the recombinant protein, and to observe its immunogenicity in mouse. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick Tie-2 was obtained by PCR from a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector PQE30, and was expressed in E. coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected into mouse subcutaneously, and the antiserum of the mouse was analyzed by ELISA and Western blot analysis. RESULTS: The recombinant protein was highly expressed in E. coli XL-1 blue, and in mouse it produced the antibody which could specifically recognize the recombinant protein. CONCLUSION: The protein of extracellular ligand binding domains of chick Tie-2 can be highly expressed in prokaryotic expression system, and the expressed protein can induce immune response in mouse. These findings are very important for the further study of this protein in anti-angiogenesis and immunotherapy research.